U.S. patent number 7,030,989 [Application Number 10/696,738] was granted by the patent office on 2006-04-18 for wavelength tunable surface plasmon resonance sensor.
This patent grant is currently assigned to University of Washington. Invention is credited to Elain S. Fu, Paul Yager.
United States Patent |
7,030,989 |
Yager , et al. |
April 18, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Wavelength tunable surface plasmon resonance sensor
Abstract
This invention provides methods, devices and device components
for sensing, imaging and characterizing changes in the composition
of a probe region. More particularly, the present invention
provides methods and devices for detecting changes in the
refractive index of a probe region positioned adjacent to a sensing
surface, preferably a sensing surface comprising a thin conducting
film supporting surface plasmon formation. In addition, the present
invention provides methods and device for generating surface
plasmons in a probe region and characterizing the composition of
the probe region by generating one or more surface plasmon
resonances curves and/or surface plasmon resonance images of the
probe region.
Inventors: |
Yager; Paul (Seattle, WA),
Fu; Elain S. (Seattle, WA) |
Assignee: |
University of Washington
(Seattle, WA)
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Family
ID: |
32230282 |
Appl.
No.: |
10/696,738 |
Filed: |
October 28, 2003 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20040130723 A1 |
Jul 8, 2004 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60421917 |
Oct 28, 2002 |
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Current U.S.
Class: |
356/445 |
Current CPC
Class: |
G01N
21/553 (20130101); G01N 21/648 (20130101) |
Current International
Class: |
G01N
21/55 (20060101) |
Field of
Search: |
;356/445,446,447,448 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 01/69209 |
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Sep 2001 |
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WO |
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WO 02/059602 |
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Aug 2002 |
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WO |
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Other References
Kovacs, G. (1982), "Optical excitation of surface
plasmon-polaritons in layered media," Electromagnetic Surface
Modes, Boardman, A.D. (ed.), John Wiley & Sons Ltd., pp
144-200. cited by other .
Naimushin, A.N. et al. (2002), "Detection of Staphylococcus aureus
enterotoxin B at femtomolar levels with a miniature integrated
two-channel surface plasmon resonance (SPR) sensor," Biosensor
Bioelectron. 17:573-584. cited by other .
Schuck, P. (1997), "Use of Surface Plasmon Resonance to Probe the
Equilibrium and Dynamic Aspects of Interactions Between Biological
Macromolecules," Annu. Rev. Biophys. Biomol. Struct. 26:541-566.
cited by other .
Welford, K. (1991), "Surface plasmon-polaritons and their uses,"
Opt. Quant. Electron. 23:1-27. cited by other .
Begley, D.L. and Seery, B.D., "Narrowband optical interference
filters" (1991) Free-Space laser communication technologies III,
SPIE Proceedings, vol. 1417:525-536. cited by other .
Berger, C. E. H., R. P. H. Kooyman, et al. (1994). "Resolution in
surface plasmon microscopy." Review of Scientific Instruments
65(9): 2829-2836. cited by other .
Berning, Peter H., (1963) "Theory and calculations of optical thin
films," Physics of Thin Films, G. Hass, New York, Academic Press
1:69-120. cited by other .
Brockman, J. M.et al. (2000), "Surface plasmon resonance imaging
measurements of ultrathin organic films." Annual Reviews of
Physical Chemistry 51:41-63. cited by other .
de Bruijn, H. E.et al. (1992), "Choice of metal and wavelength for
surface-plasmon resonance sensors: some considerations." Applied
Optics 31(4): 440-442. cited by other .
de Bruijn, H. E.et al. (1993), "Surface plasmon resonance
microscopy: improvement of the resolution by rotation of the
object." Applied Optics 32(13): 2426-2430. cited by other .
Fu, E. et al. (Jun. 2003), "Wavelength-tunable surface plasmon
resonance microscope," Rev. Sci. Instruments 74(6):3182-3184. cited
by other .
Hickel, W. and W. Knoll (1991), "Time and spatially resolved
surface plasmon optical investigation of the photodesorption of
Langmuir-Blodgett multilayer assemblies." Thin Solid Films
199:367-373. cited by other .
Hickel, W. and W. Knoll (1990), "Surface plasmon microscopy of
lipid layers." Thin Solid Films 187:349-356. cited by other .
Nelson, B.P. et al., (1999), "Near-infrared surface plasmon
resonance measurements of ultrathin films. 1. Angle shift and SPR
imaging experiments," Anal. Chem. 71(18):3928-3934. cited by other
.
Rothenhausler, B. and W. Knoll (1988), "Surface-plasmon
microscopy." Letters to Nature 332(14): 615-617. cited by
other.
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Primary Examiner: Smith; Zandra V.
Assistant Examiner: Valentin, II; Juan D.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
The work was funded through a grant by the United States government
under NIDCR grant 1UO1 DE14971-01.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority under 35 U.S.C. 119(e) to
provisional patent application 60/421,917, filed Oct. 28, 2002,
which is hereby incorporated by reference in its entirety to the
extent not inconsistent with the disclosure herein.
Claims
We claim:
1. A surface plasmon resonance sensor for sensing the refractive
index of a probe region comprising: a polychromatic light source
for generating light propagating along an incident light
propagation axis; a polarizer in optical communication with said
polychromatic light source for selecting the polarization state of
said light; an optical assembly in optical communication with said
polychromatic light source, said optical assembly comprising a
dielectric layer, a dielectric sample layer and a conducting layer
positioned between said dielectric layer and said dielectric sample
layer, wherein illumination of said optical assembly with said
light generates light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting film comprises the probe region; a
detector in optical communication with said optical assembly for
detecting said light propagating along said reflected light axis,
thereby sensing the refractive index of said probe region; and a
selectably adjustable optical interference filter positioned in the
optical path between said light source and said detector for
transmitting light having a distribution of transmitted wavelengths
selected to generate surface plasmons on a surface of said
conducting layer in contact with said dielectric sample layer,
wherein the distribution of transmitted wavelengths is continuously
tunable by adjustment of the optical interference filter, wherein
said optical interference filter is rotationally adjustable about
an axis which is orthogonal to said incident light propagation
axis, wherein rotation of said optical interference filter
selectably adjusts the tilt angle and distribution of transmitted
wavelengths of said optical interference filter.
2. The surface plasmon resonance sensor of claim 1 further
comprising a light collection and focusing element positioned
between said optical assembly and said detector, said light
collection and focusing element for collecting said light
propagating along the reflected light propagation axis and focusing
light propagating along the reflected light propagation axis onto
said detector.
3. The surface plasmon resonance sensor of claim 1 further
comprising a collimating optical element for collimating light from
said polychromatic light source, wherein said collimating optical
element is positioned between said polychromatic light element and
said optical assembly.
4. The surface plasmon resonance sensor of claim 3 where said
collimating optical element comprises a first lens, a pinhole, and
a second lens each positioned between said polychromatic light
source and said optical assembly.
5. The surface plasmon resonance sensor of claim 1 wherein said
optical interference filter is positioned between said
polychromatic light source and said optical assembly.
6. The surface plasmon resonance sensor of claim 1 wherein said
optical interference filter is a Fabry-Perot etalon.
7. The surface plasmon resonance sensor of claim 1 wherein said
optical interference filter is a linearly variable interference
filter.
8. The surface plasmon resonance sensor of claim 1 wherein rotation
of said optical interference filter selectably adjusts the center
wavelength of the distribution of transmitted wavelengths.
9. The surface plasmon resonance sensor of claim 8 wherein said
center wavelength of the distribution of transmitted wavelengths is
provided by the equation:
.lamda..function..theta..lamda..function..times..times..theta.
##EQU00006## wherein .lamda..sub.center is said center wavelength
of the distribution of transmitted wavelengths, .theta..sub.tilt is
a tilt angle, .lamda..sub.center(0) is a center wavelength at
normal incidence with respect to the reflected or incident light
propagation axes and n is the refractive index of the optical
interference filter.
10. The surface plasmon resonance sensor of claim 1 wherein said
distribution of transmitted wavelengths is characterized by a
center wavelength and said center wavelength is tunable over a
range of about 65 nm.
11. The surface plasmon resonance sensor of claim 1 wherein said
distribution of transmitted wavelengths is characterized by a
bandwidth and said bandwidth has a value selected from a range of
about 1 nm to about 100 nm.
12. The surface plasmon resonance sensor of claim 1 wherein said
detector is a charge coupled device.
13. The surface plasmon resonance sensor of claim 1 wherein said
dielectric layer has a first refractive index, wherein said
dielectric sample layer has a second refractive index which is less
than said first refractive index and wherein said light propagating
along said incident light propagation axis undergoes total internal
reflection upon interaction with said optical assembly.
14. The surface plasmon resonance sensor of claim 1 wherein said
dielectric layer is a prism.
15. The surface plasmon resonance sensor of claim 1 further
comprising a flow cell operationally connected to said optical
assembly for introducing a sample into said probe region.
16. The surface plasmon resonance sensor of claim 15 wherein said
dielectric sample layer is a sample provided by said flow cell.
17. The surface plasmon resonance sensor of claim 1 wherein said
conducting layer comprises a gold film.
18. The surface plasmon resonance sensor of claim 1 wherein said
dielectric layer and said conducting layer comprise of a
waveguide.
19. The surface plasmon resonance sensor of claim 1 wherein said
dielectric layer and said conducting layer comprise of an optical
fiber.
20. The surface plasmon resonance sensor of claim 1 comprising a
surface plasmon imaging device.
21. The surface plasmon resonance sensor of claim 1 wherein said
light source is an incoherent light source.
22. The surface plasmon resonance sensor of claim 1 further
comprising a microfluidic flow cell operationally connected to said
optical assembly for introducing a sample into said probe
region.
23. The surface plasmon resonance sensor of claim 22 wherein said
surface of said conducting layer in contact with said dielectric
sample layer comprises a side of said microfluidic flow cell.
24. The surface plasmon resonance sensor of claim 1 wherein said
surface of said conducting layer is modified to provide for
selective binding affinity.
25. The surface plasmon resonance sensor of claim 1 wherein said
surface of said conducting layer in contact with said dielectric
sample layer is modified to provide for selective adsorption
characteristics.
26. A method of sensing the refractive index of a probe region
comprising the steps of: passing light from a polychromatic light
source through a polarizer, thereby generating light propagating
along an incident light propagation axis; directing said light onto
an optical assembly, said optical assembly comprising a dielectric
layer, a dielectric sample layer and a conducting layer positioned
between said dielectric layer and said dielectric sample layer,
thereby generating light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting layer comprises said probe region;
passing said light through a selectably adjustable optical
interference filter positioned in the optical path between said
light source and a detector, wherein light having a distribution of
transmitted wavelengths is transmitted through said optical
interference filter; detecting said light having said distribution
of transmitted wavelengths with said detector, and tuning the
center wavelength of said distribution of transmitted wavelengths
by adjusting said optical interference filter to transmit light
having a continuously tunable distribution of wavelengths that
generates surface plasmons on a surface of said conducting layer in
contact with said dielectric sample layer, thereby sensing said
refractive index of said probe region, wherein said adjusting step
comprises the step of rotating said optical interference filter
about an axis which is orthogonal to said incident light
propagation axis, wherein rotation of said optical interference
filter selectably adjusts the tilt angle of said interference
filter and the distribution of wavelengths of light which are
transmitted by said interference filter.
27. The method of claim 26 wherein said adjusting step comprises
the step of systematically varying said distribution of wavelengths
transmitted by said optical interference filter.
28. The method of claim 26 wherein said adjusting step comprises
the steps of: transmitting light through said optical interference
filter having a first distribution of wavelengths, thereby
generating a first image of said probe region; transmitting light
through said optical interference filter having a second
distribution of wavelengths, thereby generating a second image of
said probe region; comparing the spectral quality of said first and
second images; and selecting a distribution of wavelengths of said
incident light which are transmitted by said optical interference
filter to enhance the spectral quality of said image.
29. The method of claim 26 wherein said optical interference filter
is positioned between said light source and said optical
assembly.
30. The method of claim 26 wherein said optical interference filter
is a Fabry-Perot etalon.
31. The method of claim 26 wherein said step of passing light
through a polarizer generates light having a p-polarization state
propagating along said incident light propagation axis.
32. The method of claim 26 where said light propagating along said
incident light propagation axis undergoes total internal reflection
upon interaction with said optical assembly.
33. The method of claim 26 further comprising the step of
collimating light from said polychromatic optical source.
34. The method claim 26 further comprising the step of focusing
said light propagating along said reflected light propagation axis
onto said detector.
35. The method of claim 26 wherein said light has wavelengths in
the near infrared region of the electromagnetic spectrum.
36. The method of claim 26 wherein said optical assembly further
comprises a flow cell operationally connected to said probe region
for delivering chemical species into said probe region.
37. The method of claim 36 further comprising the step of flowing
chemical species through said flow cell, thereby changing the
composition of said probe region.
38. The method of claim 36 further comprising the step of flowing
chemical species through said flow cell, thereby changing the
refractive index of said probe region.
39. The method of claim 36 further comprising the step of flowing
chemical species through said flow cell, thereby changing the
thickness of said probe region.
40. The method of claim 36 wherein said flow cell is a microfluidic
flow cell.
41. A method of sensing the refractive index of a probe region
comprising the steps of: passing light from a polychromatic light
source through a polarizer, thereby generating p-polarized light or
s-polarized light propagating along an incident light propagation
axis; directing said light onto an optical assembly, said optical
assembly comprising a dielectric layer, a dielectric sample layer
and a conducting layer positioned between said dielectric layer and
said dielectric sample layer, wherein light is reflected by said
optical assembly thereby generating reflected light propagating
along a reflected light propagation axis, wherein a portion of said
dielectric sample layer adjacent to said conducting layer comprises
said probe region; passing said light through an optical
interference filter positioned in the optical path between said
light source and a detector, wherein said optical interference
filter has a tilt angle with respect to said incident light
propagation axis or said reflected light propagation axis selected
so that said optical interference filter transmits incident light
having a distribution of wavelengths that generates surface
plasmons on a surface of said conducting layer in contact with said
dielectric sample layer; detecting said reflected light using said
detector, thereby measuring a first intensity of reflected light
corresponding to p-polarized light and measuring a second intensity
of reflected light corresponding reflected s-polarized light;
calculating an observed percent reflectivity by determining the
ratio of said first intensity of reflected light to said second
intensity of reflected light; determining a correction factor by
measuring the ratio of the intensity of p-polarized light
transmitted by said optical interference filter to s-polarized
light transmitted by said optical interference filter having said
tilt angle; and calculating a percent reflectivity corrected for
polarization dependent transmission of light transmitted by said
optical interference filter by dividing said observed percent
reflectivity by said correction factor, thereby sensing the
refractive index of said probe region.
42. The method of claim 41 further comprising the steps of:
determining a plurality of correction factors corresponding to
different tilt angles by measuring the ratios of the intensity of
p-polarized light transmitted by said optical interference filter
to s-polarized light transmitted by said optical interference
filter having a plurality of tilt angles; plotting said correction
factors as a function of tilt angle, thereby generating a
calibration plot; fitting a curve to said calibration plot, thereby
generating a calibration curve; and determining said correction
factor using said calibration curve.
43. The method of claim 41 where said step of determining said
correction factor by measuring the ratio of the intensity of
p-polarized light transmitted by said optical interference filter
to s-polarized light transmitted by said optical interference
filter having said tilt angle is carried out by separately
measuring the intensities of p-polarized light and s-polarized
light passed by said interference filter in the absence of surface
plasmon formation.
44. A method of generating an image of a probe region comprising
the steps of: passing light from a polychromatic light source
through a polarizer, thereby generating p-polarized light or
s-polarized light propagating along an incident light propagation
axis; directing said light onto an optical assembly, said optical
assembly comprising a dielectric layer, a dielectric sample layer
and a conducting layer positioned between said dielectric layer and
said dielectric sample layer, wherein light is reflected by said
optical assembly thereby generating reflected light propagating
along a reflected light propagation axis, wherein a portion of said
dielectric sample layer adjacent to said conducting layer comprises
said probe region; passing said light through an optical
interference filter positioned in the optical path between said
light source and a detector, wherein said optical interference
filter has a tilt angle with respect to said incident light
propagation axis or said reflected light propagation axis selected
so that said optical interference filter transmits incident light
having a distribution of wavelengths that generates surface
plasmons on a surface of said conducting layer in contact with said
dielectric sample layer; detecting said reflected light using said
detector, thereby measuring a first two-dimensional distribution of
reflected light intensities corresponding to p-polarized light and
measuring second two-dimensional distribution of reflected light
intensities corresponding to s-polarized light; calculating a two
dimensional distribution of observed percent reflectivities by
determining the ratios of p-polarized reflected light intensities
in said first two-dimensional distribution to s-polarized light
intensities in said second two-dimensional distributions of
reflected light intensities; determining a two-dimensional array of
correction factors corresponding to said tilt angle by measuring
the ratios of the intensity of p-polarized light transmitted by
said optical interference filter to s-polarized light transmitted
by said optical interference filter having said tilt angle for each
element in said two dimensional distribution of reflected light
intensities ; and calculating a two dimensional distribution of
percent reflectivities corrected for polarization dependent
transmission of light transmitted by said optical interference
filter by dividing said observed percent reflectivities by said
correction factors in said two dimensional array, thereby
generating an image of said probe region.
45. The method of claim 44 further comprising the steps of:
determining a plurality of two dimensional arrays of correction
factors corresponding to different tilt angles by measuring the
ratios of the intensity of p-polarized light transmitted by said
optical interference filter to s-polarized light transmitted by
said optical interference filter at a plurality of tilt angles;
plotting said correction factors as a function of tilt angle,
thereby generating a plurality of calibration plots; fitting curves
to said calibration plots, thereby generating a plurality of
calibration curves; and determining said correction factors using
said plurality of said calibration curves.
46. The method of claim 44 further comprising the step of
optimizing the contrast of said image of said probe region by
varying the center wavelength of said distribution of transmitted
wavelength by rotating said optical interference filter about an
axis which is orthogonal to said incident light propagation axis or
said reflected light propagation axis.
47. The method of claim 44 where said step of determining a
two-dimensional array of correction factors corresponding to said
tilt angle by measuring the ratios of the intensity of p-polarized
light transmitted by said optical interference filter to
s-polarized light transmitted by said optical interference filter
having said tilt angle for each element in said two dimensional
distribution of reflected light intensities is carried out by
separately measuring the intensities of p-polarized light and
s-polarized light passed by said interference filter in the absence
of surface plasmon formation.
48. A surface plasmon resonance sensor for sensing the refractive
index of a probe region comprising: a polychromatic light source
for generating light propagating along an incident light
propagation axis; a polarizer in optical communication with said
polychromatic light source for selecting the polarization state of
said light; an optical assembly in optical communication with said
polychromatic light source, said optical assembly comprising a
dielectric layer, a dielectric sample layer and a conducting layer
positioned between said dielectric layer and said dielectric sample
layer, wherein illumination of said optical assembly with said
light generates light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting film comprises the probe region; a
detector in optical communication with said optical assembly for
detecting said light propagating along said reflected light axis,
thereby sensing the refractive index of said probe region; and an
optical interference filter positioned in the optical path between
said light source and said detector for transmitting light having a
distribution of transmitted wavelengths selected to generate
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, wherein the distribution of
transmitted wavelengths is continuously tunable by adjustment of
the optical interference filter, wherein said optical interference
filter is rotationally adjustable about an axis which is orthogonal
to said reflected light propagation axis, wherein rotation of said
optical interference filter selectably adjusts the tilt angle and
distribution of transmitted wavelengths of said optical
interference filter.
49. The surface plasmon resonance sensor of claim 48 wherein said
optical interference filter has first and second substantially
parallel ends and said first end has a tilt angle selected over a
range of 0.degree. to about 35.degree..
50. The surface plasmon resonance sensor of claim 48 wherein said
distribution of transmitted wavelengths is characterized by a
center wavelength and said center wavelength is tunable over a
range of about 65 nm.
51. The surface plasmon resonance sensor of claim 48 wherein said
distribution of transmitted wavelengths is characterized by a
bandwidth and said bandwidth has a value selected from a range of
about 1 nm to about 100 nm.
52. A surface plasmon resonance sensor for sensing the refractive
index of a probe region comprising: a polychromatic light source
for generating light propagating along an incident light
propagation axis; a polarizer in optical communication with said
polychromatic light source for selecting the polarization state of
said light; an optical assembly in optical communication with said
polychromatic light source, said optical assembly comprising a
dielectric layer, a dielectric sample layer and a conducting layer
positioned between said dielectric layer and said dielectric sample
layer, wherein illumination of said optical assembly with said
light generates light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting film comprises the probe region; a
detector in optical communication with said optical assembly for
detecting said light propagating along said reflected light axis,
thereby sensing the refractive index of said probe region; and an
optical interference filter positioned in the optical path between
said light source and said detector for transmitting light having a
distribution of transmitted wavelengths selected to generate
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, wherein the distribution of
transmitted wavelengths is continuously tunable by adjustment of
the optical interference filter, wherein said optical interference
filter is rotationally adjustable about an axis which is orthogonal
to said incident light propagation axis, wherein rotation of said
optical interference filter selectably adjusts the distribution of
wavelengths that are substantially prevented from transmitting
through said optical interference filter.
53. The surface plasmon resonance sensor of claim 52 wherein said
optical interference filter has first and second substantially
parallel ends and said first end has a tilt angle selected over a
range of 0.degree. to about 35.degree..
54. The surface plasmon resonance sensor of claim 52 wherein said
distribution of transmitted wavelengths is characterized by a
center wavelength and said center wavelength is tunable over a
range of about 65 nm.
55. The surface plasmon resonance sensor of claim 52 wherein said
distribution of transmitted wavelengths is characterized by a
bandwidth and said bandwidth has a value selected from a range of
about 1 nm to about 100 nm.
56. A surface plasmon resonance sensor for sensing the refractive
index of a probe region comprising: a polychromatic light source
for generating light propagating along an incident light
propagation axis; a polarizer in optical communication with said
polychromatic light source for selecting the polarization state of
said light; an optical assembly in optical communication with said
polychromatic light source, said optical assembly comprising a
dielectric layer, a dielectric sample layer and a conducting layer
positioned between said dielectric layer and said dielectric sample
layer, wherein illumination of said optical assembly with said
light generates light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting film comprises the probe region; a
detector in optical communication with said optical assembly for
detecting said light propagating along said reflected light axis,
thereby sensing the refractive index of said probe region; and an
optical interference filter positioned in the optical path between
said light source and said detector for transmitting light having a
distribution of transmitted wavelengths selected to generate
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, wherein the distribution of
transmitted wavelengths is continuously tunable by adjustment of
the optical interference filter, wherein said optical interference
filter is rotationally adjustable about an axis which is orthogonal
to said reflected light propagation axis, wherein rotation of said
optical interference filter selectably adjusts the distribution of
wavelengths that are substantially prevented from transmitting
through said optical interference filter.
57. The surface plasmon resonance sensor of claim 56 wherein said
optical interference filter has first and second substantially
parallel ends and said first end has a tilt angle selected over a
range of 0.degree. to about 35.degree..
58. The surface plasmon resonance sensor of claim 56 wherein said
distribution of transmitted wavelengths is characterized by a
center wavelength and said center wavelength is tunable over a
range of about 65 nm.
59. The surface plasmon resonance sensor of claim 56 wherein said
distribution of transmitted wavelengths is characterized by a
bandwidth and said bandwidth has a value selected from a range of
about 1 nm to about 100 nm.
60. A surface plasmon resonance sensor for sensing the refractive
index of a probe region comprising: a polychromatic light source
for generating light propagating along an incident light
propagation axis; a polarizer in optical communication with said
polychromatic light source for selecting the polarization state of
said light; an optical assembly in optical communication with said
polychromatic light source, said optical assembly comprising a
dielectric layer, a dielectric sample layer and a conducting layer
positioned between said dielectric layer and said dielectric sample
layer, wherein illumination of said optical assembly with said
light generates light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting film comprises the probe region; a
detector in optical communication with said optical assembly for
detecting said light propagating along said reflected light axis,
thereby sensing the refractive index of said probe region; and an
optical interference filter positioned in the optical path between
said light source and said detector for transmitting light having a
distribution of transmitted wavelengths selected to generate
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, wherein the distribution of
transmitted wavelengths is continuously tunable by adjustment of
the optical interference filter, wherein said optical interference
filter has first and second substantially parallel ends and said
first end has a tilt angle selected over the range of 0.degree. to
about 35.degree..
61. The surface plasmon resonance sensor of claim 60 wherein said
optical interference filter has first and second substantially
parallel ends and said first end has a tilt angle selected over a
range of 0.degree. to about 35.degree..
62. The surface plasmon resonance sensor of claim 60 wherein said
distribution of transmitted wavelengths is characterized by a
center wavelength and said center wavelength is tunable over a
range of about 65 nm.
63. The surface plasmon resonance sensor of claim 60 wherein said
distribution of transmitted wavelengths is characterized by a
bandwidth and said bandwidth has a value selected from a range of
about 1 nm to about 100 nm.
64. A method of sensing the refractive index of a probe region
comprising the steps of: passing light from a polychromatic light
source through a polarizer, thereby generating light propagating
along an incident light propagation axis; directing said light onto
an optical assembly, said optical assembly comprising a dielectric
layer, a dielectric sample layer and a conducting layer positioned
between said dielectric layer and said dielectric sample layer,
thereby generating light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting layer comprises said probe region;
passing said light through an optical interference filter
positioned in the optical path between said light source and a
detector, wherein light having a distribution of transmitted
wavelengths is transmitted through said optical interference
filter; detecting said light having said distribution of
transmitted wavelengths with said detector, and tuning the center
wavelength of said distribution of transmitted wavelengths by
adjusting said optical interference filter to transmit light having
a continuously tunable distribution of wavelengths that generates
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, thereby sensing said refractive
index of said probe regions, wherein said adjusting step comprises
the step of rotating said optical interference filter about an axis
which is orthogonal to said reflected light propagation axis,
wherein rotation of said optical interference filter selectably
adjusts the tilt angle of said interference filter and the
distribution of wavelengths of light which are transmitted by said
interference filter.
65. The method of claim 64 wherein said adjusting step comprises
the steps of: transmitting light through said optical interference
filter having a first distribution of wavelengths, thereby
generating a first image of said probe region; transmitting light
through said optical interference filter having a second
distribution of wavelengths, thereby generating a second image of
said probe region; comparing the spectral quality of said first and
second images; and selecting a distribution of wavelengths of said
incident light which are transmitted by said optical interference
filter to enhance the spectral quality of said image.
66. The method of claim 64 wherein said adjusting step comprises
the step of systematically varying said distribution of wavelengths
transmitted by said optical interference filter.
67. A method of sensing the refractive index of a probe region
comprising the steps of: passing light from a polychromatic light
source through a polarizer, thereby generating light propagating
along an incident light propagation axis; directing said light onto
an optical assembly, said optical assembly comprising a dielectric
layer, a dielectric sample layer and a conducting layer positioned
between said dielectric layer and said dielectric sample layer,
thereby generating light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting layer comprises said probe region;
passing said light through an optical interference filter
positioned in the optical path between said light source and a
detector, wherein light having a distribution of transmitted
wavelengths is transmitted through said optical interference
filter; detecting said light having said distribution of
transmitted wavelengths with said detector, and tuning the center
wavelength of said distribution of transmitted wavelengths by
adjusting said optical interference filter to transmit light having
a continuously tunable distribution of wavelengths that generates
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, thereby sensing said refractive
index of said probe region, wherein said adjusting step comprises
the step of rotating said optical interference filter about an axis
which is orthogonal to said incident light propagation axis,
wherein rotation of said optical interference filter selectably
adjusts the tilt angle of said interference filter and the
distribution of wavelengths that are substantially prevented from
transmitting through said optical interference filter.
68. The method of claim 67 wherein said adjusting step comprises
the steps of: transmitting light through said optical interference
filter having a first distribution of wavelengths, thereby
generating a first image of said probe region; transmitting light
through said optical interference filter having a second
distribution of wavelengths, thereby generating a second image of
said probe region; comparing the spectral quality of said first and
second images; and selecting a distribution of wavelengths of said
incident light which are transmitted by said optical interference
filter to enhance the spectral quality of said image.
69. The method of claim 67 wherein said adjusting step comprises
the step of systematically varying said distribution of wavelengths
transmitted by said optical interference filter.
70. A method of sensing the refractive index of a probe region
comprising the steps of: passing light from a polychromatic light
source through a polarizer, thereby generating light propagating
along an incident light propagation axis; directing said light onto
an optical assembly, said optical assembly comprising a dielectric
layer, a dielectric sample layer and a conducting layer positioned
between said dielectric layer and said dielectric sample layer,
thereby generating light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting layer comprises said probe region;
passing said light through an optical interference filter
positioned in the optical path between said light source and a
detector, wherein light having a distribution of transmitted
wavelengths is transmitted through said optical interference
filter; detecting said light having said distribution of
transmitted wavelengths with said detector, and tuning the center
wavelength of said distribution of transmitted wavelengths by
adjusting said optical interference filter to transmit light having
a continuously tunable distribution of wavelengths that generates
surface plasmons on a surface of said conducting layer in contact
with said dielectric sample layer, thereby sensing said refractive
index of said probe region, wherein said adjusting step comprises
the step of rotating said optical interference filter about an axis
which is orthogonal to said reflected light propagation axis,
wherein rotation of said optical interference filter selectably
adjusts the tilt angle of said interference filter and the
distribution of wavelengths that are substantially prevented from
transmitting through said optical interference filter.
71. The method of claim 70 wherein said adjusting step comprises
the steps of: transmitting light through said optical interference
filter having a first distribution of wavelengths, thereby
generating a first image of said probe region; transmitting light
through said optical interference filter having a second
distribution of wavelengths, thereby generating a second image of
said probe region; comparing the spectral quality of said first and
second images; and selecting a distribution of wavelengths of said
incident light which are transmitted by said optical interference
filter to enhance the spectral quality of said image.
Description
BACKGROUND OF THE INVENTION
Surface plasmon resonance (SPR) microscopy is a technique that uses
excitation of surface plasmons (SPs) to detect chemical and
physical changes in a probed region adjacent to a sensing surface.
A variety of sensors based on SPR techniques have been developed
which provide a sensitive means of characterizing the thickness and
index of refraction of ultrathin films occurring at the surface of
a thin metal film. In recent years, SP sensors have been used
extensively to characterize chemical and physical properties of a
variety of biological materials and to probe binding events in real
time. For example, SP sensors have been used successfully to
characterize the morphology of a range of surfaces, probe the
kinetics and dynamics of interactions between proteins, proteins
and DNA and proteins and small molecules, monitor antibody-antigen
binding and characterize DNA hybridization processes.
Surface plasmons, also know as surface plasmon waves or plasmon
polaritons, are charge density waves, which propagate parallel to
an interface between a conducting or semiconducting thin film and a
dielectric sample layer. SPs are generated by coupling radiant
energy from incident photons into the oscillating modes of free
electrons present in a conducting material, such as a metal, or
semiconductor material. SPs are highly localized at the surface of
the conducting (or semiconducting) layer and the intensity of the
electric field of a SP decays exponentially in directions
perpendicular to the plane in which it propagates. The spatial
distribution of a SP may be quantitatively described by a
characteristic decay length corresponding to the distance over
which the intensity of the SP decays to e.sup.-1 times its value at
the conductor (or semiconductor)--dielectric sample layer
interface. Decay length (L) is provided by the expression: .times.
.times..function. ##EQU00001## wherein Re refers to the real part
of the quantity in parentheses, k.sub.sp is the surface plasmon
wavevector and k.sub.s is the wavevector in the dielectric sample
layer adjacent to the conductor (or semiconductor). For a
dielectric sample layer comprising water and a conducting thin film
comprising gold the decay length is equal to about 83.1 nm for
light having a wavelength of about 632.8 nm. The highly localized
nature of SPs make them ideally suited for detecting very small
changes in refractive index occurring in sensing regions proximate
to a sensing surface (.ltoreq. about 300 nm).
In conventional SPR methods, a SP is excited by evanescent
electromagnetic waves generated upon total internal reflection of
an incident light beam. In the Kretschmann-Raether geometry,
evanescent electromagnetic waves penetrate a thin metal film
(.apprxeq.50 nm) positioned between higher and lower refractive
index dielectric layers and excite a SP, which propagates parallel
to the outer surface of the metal film adjacent to the lower
refractive index layer. The prism is needed to achieve the
wavevector matching condition between the incident excitation light
and the surface plasmons. For a given dielectric sample, photons of
a certain wavelength and incident at a certain angle will generate
evanescent waves that penetrate the metal layer and excite surface
plasmons at the metal-dielectric sample interface. The intensity of
reflected light will therefore be reduced and can be monitored as a
signal of SP generation. Alternatively, in the Otto SPR
configuration, the metal layer and prism are separated by an air
gap and SPs are excited on the side of the metal film adjacent to
the prism. A drawback of the Otto SPR configuration is that it is
experimentally difficult to maintain a very thin and constant
thickness air gap. Finally, in other SPR methods, surface plasmons
are created by evanescent fields generated as light propagates down
a fiber optic or waveguide having a thin metal interior layer
Excitation of SPs via total internal reflection is a resonant
phenomenon that depends on the wavevector of the incident light
(i.e. both the wavelength and angle of incidence of the incident
light beam. In addition, excitation of SPs is dependent on the
indices of refraction and thickness of the higher refractive index
layer, lower refractive index sample layer and conducting (or
semiconducting) thin film used to couple radiant energy into the
oscillating modes of free electrons present in the conductor. The
dispersion equation for a SP is provided by the equation: .times.
.times..times..times. ##EQU00002## wherein k.sub.0 is the free
space wavevector (k.sub.0=.omega./c); .di-elect cons..sub.c and
.di-elect cons..sub.d are the complex permittivities of the
conducting (or semiconducting) thin film and the lower refractive
index dielectric sample layer, respectively and .omega. is the
angular frequency. A resonance condition of exciting an SP is that
the parallel component of the incident wavevector (k.sub.par), must
equal the surface plasmon wave vector (k.sub.sp):
k.sub.par=k.sub.sp (III). The parallel component of the incident
wavevector may be expressed in terms of the index of refraction of
the medium in which the light is incident, n, the angle of
incidence, .theta., and the wavelength of the incident light beam,
.lamda., by the equation for formation of a SP: .times.
.times..times. .times..times. .times..pi..times. .times..times.
.times..function..theta..lamda. ##EQU00003## Substituting equations
II and IV into equation III provides the following relationship
expressing the resonance condition for the formation of a surface
plasmon in terms of the angle of incidence and wavelength of the
incident beam: .times. .times..pi..times. .times..times.
.times..function..theta..lamda..times..times. ##EQU00004## As is
evident from equation V, for a given metal film thickness and set
of refractive indices of dielectric layers, the resonance condition
may be satisfied by variation of either the angle of incidence or
the wavelength of the incident light beam, or both.
In the derivation of the dispersion relation for the SP, equation
II, two additional conditions that must be satisfied for surface
plasmon generation to occur become apparent. First, SPs are
p-polarized and so can only be excited by p-polarized incident
light. And second, SPs are only supported at an interface made up
of media with real permittivites of opposite sign.
As illustrated by equations II-V, changes in the refractive index
of the dielectric sample layer adjacent to the thin metal film
changes the resonance condition for generating a SP. This change in
resonance condition may be monitored directly by measuring the
intensity of the reflected incident beam as a function of angle of
incidence, wavelength of the incident beam or both. Satisfaction of
the resonance condition results in a sharp attenuation in the
intensity of the reflected beam caused by a conversion of radiant
energy of the incident beam into SPs at the interface between the
thin metal film and the lower refractive index layer. Due to their
spatially localized nature, SPs have also been used to excite
photoluminescent materials. Specifically, energy from a SP is
coupled to a photoluminescent material in a manner resulting in
excitation of an electronic transition providing fluorescence or
photoluminescence. An additional detector can be positioned in
optical communication with the sensing surface to measure the
intensity of fluorescence of materials pumped by the SPR process.
The combination of attenuated reflectance SPR methods and SPR
induced fluorescence has been demonstrated to provide a sensitive
means of characterizing chemical and physical changes occurring at
a senor surface.
Sensors based on SPR utilize the dependence of the SPR resonance
condition on changes in the refractive index of a lower refractive
index dielectric sample layer positioned adjacent to the thin metal
(or semiconductor) film. In typical sensing applications, changes
in the resonance condition for formation of SPs are monitored in
real time and directly related to chemical or physical changes
occurring at a sensing surface adjacent to the thin metal (or
semiconductor) film. Sensors based on SPR may provide selective
detection of materials and compounds by manipulating the chemical
or physical properties of the sensing surface. In these
applications, the sensing surface may be coated with a material
exhibiting selective binding characteristics such that the
refractive index varies in the presence of a specific material to
be sensed. For example, the sensing surface may be made sensitive
to a particular antibody by coating it with an antigen to that
antibody. Using these principles, SPR detection has been
successfully incorporated into a number of commercially available
biological sensing devices including the sensors and screening
devices manufactured by BIAcore, Inc.
Generally, a SPR optical configuration comprises (1) a source of
electromagnetic radiation, (2) an optically transmissive component
having a first refractive index, (3) a dielectric sample layer (or
probe region) having a second refractive index less than that of
the first refractive index of the optically transmissive component,
(4) a conducting or semiconducting thin film positioned between the
optically transmissive component and the dielectric sample layer
(probe region) and (5) a detector. In this configuration, an
incident beam is transmitted through the transparent region at an
angle of incidence such that it undergoes total internal reflection
at the interface between the optical transmissive component and the
conducting thin film. The reflected incident beam is collected and
directed to a detector capable of measuring its intensity as
function of time. If the resonance condition outlined in Equations
II to V is met, radiant energy is converted into a SP at the
interface between the conducting or semiconducting thin film and
the dielectric sample layer resulting in a measurable decrease in
the intensity of the reflected incident beam.
Sensors based on SPR may utilize a number of different optical
configurations. Exemplary optical configurations are described in
Rothenhausler, B. and W. Knoll (1988). "Surface-plasmon
microscopy." Letters to Nature 332(14): 615-617., Hickel, W. and W.
Knoll (1990). "Surface plasmon microscopy of lipid layers." Thin
Solid Films 187: 349-356, Hickel, W. and W. Knoll (1991). "Time and
spatially resolved surface plasmon optical investigation of the
photodesorption of Langmuir-Blodgett multilayer assemblies." Thin
Solid Films 199: 367-373, de Bruijn, H. E., R. P. H. Kooyman, et
al. (1992), "Choice of metal and wavelength for surface-plasmon
resonance sensors; some considerations." Applied Optics 31(4):
440-442, de Bruijn, H. E., R. P. H. Kooyman, et al. (1993).
"Surface plasmon resonance microscopy; improvement of the
resolution by rotation of the object." Applied Optics 32(13):
2426-2430, Berger, C. E. H., R. P. H. Kooyman, et al. (1994).
"Resolution in surface plasmon microscopy." Review of Scientific
Instruments 65(9): 2829-2837 and Brockman, J. M., B. P. Nelson, et
al. (2001) "Surface plasmon resonance imaging measurements of
ultrathin organic films." Annual Reviews of Physical Chemistry
51(1): 41-47, which are hereby incorporated by reference in their
entireties to the extent not inconsistent with the present
application.
The most common configuration in SPR sensing applications involves
angle modulation of a substantially monochromatic, coherent
incident light beam. In this technique, a surface plasmon resonance
curve is generated by measuring the intensity of a reflected,
substantially monochromatic, coherent incident beam, as the angle
of incidence is systematically varied. Satisfaction of the SP
resonance condition results in a measurable attenuation of the
intensity of the incident beam corresponding to the minimum of a
curve of reflected beam intensity versus incident angle. The angle
corresponding to this minimum, referred to as the resonant angle
(.theta..sub.sp), is dependent on the index of refraction near the
surface of the conducting layer. Adsorption or binding of materials
in the sensing region adjacent to the conducting layer changes the
index of refraction in the sensing region and causes a measurable
shift in the value of .theta..sub.sp. Quantification of the shift
in .theta..sub.sp, therefore, provides a sensitive means of
observing and characterizing changes in the composition and
concentration of materials in sensing region. For example, studies
have demonstrated linear correlations exist between resonance angle
shifts and protein concentrations in the sensing region.
Despite the demonstrated effectiveness of angle modulation SPR
techniques, theses optical configurations have several practical
limitations. First, angle modulation optical configurations require
use of complicated optical component rotation assemblies for
selectably adjusting the angle of incidence of the incident beam.
Typically, such assemblies provide for rotation of a combination of
a light source, beam shaping optics and polarizing optics and/or
rotation of a combination of light collection optics and a
detector. Optical configurations requiring use of such complex
rotation assemblies are undesirable because they are costly,
spatially restrictive and require frequent maintenance and
realignment. Second, use of complex optical component rotation
assembles increases an instrument's sensitivity to optical
misalignment caused by vibration and variations in ambient
temperature and pressure. Finally, use of coherent light sources,
such as lasers, in angle modulation SPR techniques results in
unwanted optical interference of reflected beam components. Such
optical interference is undesirable because it results in fringe
patterns, which substantially degrades the optical quality of
images obtained by SPR techniques.
Another optical configuration common to SPR sensing applications
involves wavelength modulation. In wavelength modulation optical
configurations, the intensity of the reflected incident beam is
monitored for a fixed angle of incidence as the wavelength of the
incident beam is systematically varied. In these techniques, a
surface plasmon resonance curve is generated by measuring the
intensity of a reflected incident beam, as the wavelength of the
incident beam is varied. The wavelength corresponding to the
minimum of a curve of reflected beam intensity verse wavelength,
referred to as the resonant wavelength (.lamda..sub.sp), indicates
satisfaction of the resonance condition and is dependent on the
index of refraction of a sensing region adjacent to the surface of
the conducting layer. Quantification of the shift in
.lamda..sub.sp, therefore, provides a sensitive means of observing
and characterizing changes in the composition and concentration of
materials in sensing region. SPR wavelength modulation techniques
commonly employ a constant angle of incidence and, therefore, do
not require use of bulky optical rotation assembles.
Another application of SPR to sensing is SPR imaging techniques,
wherein spatial differences in the reflectivity of an incident beam
are measured as a function of time. In this technique, a
collimated, monochromatic light beam is used for excitation of SPs
and reflected light corresponding to a probe region is monitored by
a two-dimensional array detector, such as a charge coupled device
or camera. Differences in composition in the probe region are
monitored in real time by observing a two-dimensional distribution
of measured reflected light intensities. The thickness and
refractive index of materials absorbed or bound to certain regions
of the probe area may satisfy the SP resonance condition and
provide for efficient SP formation. Therefore, these regions will
exhibit attenuated reflected light intensities. Other regions of
the probe area, in contrast, may comprise absorbed or bound
materials having refractive indices which do not satisfy the SP
resonance condition and do not result in efficient SP formation.
Therefore, these regions will exhibit high reflectivities of the
incident beam. Differences in the reflectivities of regions having
different chemical and physical properties result in an image
characterizing the entire probe area. The maximum contrast between
regions in the probe area can be obtained by varying the imaging
angle or wavelength of the SPR system.
Brockman, J. M., B. P. Nelson, et al. (2001). "Surface plasmon
resonance imaging measurements of ultrathin organic films." Annual
Reviews of Physical Chemistry 51(1): 41-47 describes an optical
configuration that is reported to improve quality and sensitivity
of images generated by SPR imaging techniques. The authors disclose
an optical arrangement comprising a collimated white light source,
polarizer, prism--thin gold film sample assembly, narrow band
interference filter and charge couple device (CCD) camera. The
reference shows five SPR images corresponding to five different
interference filters, which passes different wavelengths of
excitation light. Although the authors report that SPR image
quality may be optimized by selection of an interference filter
having the appropriate transmission characteristics, the disclosed
methods require time consuming, iterative image quality adjustment
by manual removal and insertion of different interference filters.
The authors principally depend on angle scanning to optimally
contrast the samples in their probe region. Moreover, removal and
insertion of optical interference filters requires repeated
alignment of the excitation and detection optical arrangements. In
addition, the teaching of the reference is limited to optical
configurations providing discrete detection wavelength selection
and does not provide the ability to tune the excitation or
detection wavelength over a continuous range of values. Finally,
the methods disclosed expose the sample to significant intensities
of light having wavelengths not detected by the CCD camera, which
do not contribute to SPR image formation and may damage materials
in the probe region.
It will be appreciated from the foregoing that a clear need exists
for methods and devices for generating SPs in thin conducting (or
semiconducting films) which do not utilize angle modulation SPR,
particularly angle modulation SPR optical configurations having
complex rotational assemblies. Further, methods and devices for
wavelength modulation SPR sensing and/or imaging having
continuously tunable, incoherent light sources are needed. Finally,
tunable SPR instruments are needed which eliminate undesirable
optical interference problems and provide enhanced sensitivity and
resolution.
SUMMARY OF THE INVENTION
This invention provides methods, devices and device components for
sensing, imaging and characterizing changes in the composition of a
probe region. More particularly, the present invention provides
methods and devices for detecting changes in the refractive index
of a probe region positioned adjacent to a sensing surface,
preferably a sensing surface comprising a thin conducting film
supporting SP formation. In addition, the present invention
provides methods and device for generating surface plasmons in a
probe region and characterizing the composition of the probe region
by generating one or more surface plasmon resonances curves and/or
surface plasmon resonance images of the probe region. The methods
and devices of the present invention may be used to detect and
characterize adsorption, absorption or binding of chemical species,
such as molecules and ions, to a probe region, particularly a probe
region having selected binding affinity and/or other selected
chemical or physical properties. Further, the present invention
provides wavelength tunable SPR sensing devices and imaging devices
that are capable of sensing changes in the occurrence of SPR and/or
the SP resonant wavelength required for SPR formation as a function
of time, particularly with respect to a probe region undergoing
physical and/or chemical changes. Wavelength tunable SPR sensing
devices of the present invention may be used to detect species in a
solution that are in contact with or near the sensing surface.
It is an object of the present invention to provide tunable SPR
sensing and imaging devices that do not require angular modulation,
particularly devices which do not require complex optical rotation
assemblies for varying the angle of incidence in conventional
angular modulation SPR optical configurations. It is further an
object of the present invention to provide methods and devices
which minimize the occurrence of optical interference, particularly
methods and devices which eliminate the occurrence of fringe
patterns and speckle which degrade SPR image quality and obfuscate
SPR sensing measurements. It is yet another object of the present
invention to provide methods and devices for detecting materials,
such as atoms, molecules, ions or aggregates of atoms, molecules
and ions, which do not require pre-detection labeling processes,
such as fluorescent labeling or radioactive labeling processes.
In one aspect, the present invention provides a wavelength tunable
surface plasmon resonance sensor for sensing, monitoring and
characterizing changes in the refractive index of a probe region.
Wavelength tunable surface plasmon resonance sensors of the present
invention provide excitation light and/or detected light having a
distribution of wavelengths that is selectably adjustable. An
exemplary wavelength tunable surface plasmon resonance sensor
comprises an incoherent, polychromatic light source, a polarizer,
an SPR optical assembly, a detector and a selectably adjustable
wavelength selector. In these embodiments, the polychromatic light
source is positioned in optical communication with the polarizer
and SPR optical assembly. Light generated by the light source
propagates along an incident light propagation axis and is directed
through a polarizer resulting in light having a selected
polarization orientation, preferably substantially p-polarized
light or s-polarized light. Preferred polarizers of the present
invention provide a means of easily switching between incident
light having a p-polarization orientation and s-polarization
orientation. Light having a selected polarization orientation is
directed onto the SPR optical assembly. In an exemplary embodiment,
the SPR optical assembly comprises a dielectric layer, a dielectric
sample layer and a conducting layer position between the dielectric
layer and the dielectric sample layer. The dielectric sample layer
adjacent to the conducting film comprises the probe region.
Exemplary SPR sensors of the present invention further comprise one
or more optical collimation elements positioned between the
polychromatic light source and the SPR optical assemble for
collimating the light beam directed to the SPR optical
assembly.
Illumination of the SPR optical assembly at angles of incidence
resulting in total internal reflection generates light propagating
along a reflected light propagation axis. In exemplary
configurations, light propagating along the incident light
propagation axis or light propagating along the reflected light
axis is passed through a selectably adjustable wavelength selector
positioned in the optical path between the light source and the
detector. In a preferred embodiment, the selectably adjustable
wavelength selector transmits light having a distribution of
transmitted wavelengths selected to generate surface plasmons on
the surface of the conducting layer adjacent to the probe region.
Light propagating along the reflected light axis is detected by the
detector, thereby sensing the refractive index of the probe region.
Optionally, light propagating along the reflected light axis may be
collected by a light collection element and focused onto the
detector to improve detection sensitivity and resolution.
In one embodiment, the present invention provides a means of
quantifying percentage reflectivities of p-polarized incident light
that is reflected from the SPR optical assembly. In an exemplary
embodiment, the SPR optical assembly is alternately illuminated
with p-polarized light and s-polarized light by selective
adjustment of the polarizer. Illumination of the SPR optical
assembly with p-polarized light having a wavelength satisfying the
SP resonance condition converts radiant energy to SPs, which
decreases the intensity of the reflected p-polarized light. Because
s-polarized light does not result in SP formation, decreases in the
intensity of reflected p-polarized light may be accurately
characterized in terms of a percentage reflectivity by comparing
the intensities of detected p-polarized light and s-polarized light
resulting from alternative illumination of the SPR optical assembly
with substantially p-polarized and s-polarized light beams.
In the present invention, the selectably adjustable wavelength
selector provides wavelength tuning functionality useful for
characterizing SP resonance conditions and measuring a resonant
wavelength necessary for SP formation. Further, the selectably
adjustable wavelength selector of the present invention eliminates
the need for angular modulation for sensing changes in the
refractive index of a probe region by SPR methods. In the context
of this aspect of the present invention, wavelength tuning refers
to selective variation of incident and/or detected light in a
manner satisfying SP resonance conditions and resulting in SP
excitation. As SP resonance conditions are a dependent on the
refractive index of the probe region, wavelength tunable SPR
sensors of the present invention provide a means of detecting and
monitoring physical and chemical properties, such as composition,
binding affinity and reactivity, of the probe region.
Preferred wavelength selectors provide a distribution of
transmitted wavelengths that is selectably adjustable. The
distribution of transmitted wavelengths of light of the present
invention may be characterized in terms of a center wavelength,
bandwidth and wavelength intensity profile. Exemplary wavelength
selectors of the present invention are capable of selectively
adjusting the center wavelength of a distribution of transmitted
wavelengths over a continuum of values. In the present invention,
the center wavelength, bandwidth and/or wavelength intensity
profile of light transmitted by the wavelength selector may be
selected to enhance the sensitive or resolution of a SPR sensing
measurement. Alternatively, the center wavelength of the
distribution, bandwidth and/or wavelength intensity profile of
light transmitted by the wavelength selector may be selected to
enhance SPR image quality (i.e. optimal refractive index contrast
within different areas of the probe region).
To observe SP formation, characterize the SP resonance condition or
measure the SP resonant wavelength, exemplary SPR sensors of the
present invention monitor a decrease in the intensity of light
reflected from the SPR optical assembly. Selectably adjustable
wavelength selectors of the present invention provide a means of
adjusting the wavelengths of light that are detected. This allows
resonant wavelengths to be accurately measured and also allows for
characterization of a SP resonance curve by measuring reflected
light intensities as a function of the wavelength of light
transmitted by the wavelength selector. The ability to selectively
adjust the wavelengths of light that are detected provides this
function of the wavelength tunable SPR sensors of the present
invention. Therefore, selectably adjustable wavelength selectors of
the present invention may be positioned anywhere in the optical
path of collimated light from the polychromatic light source to the
detector. In one embodiment, the selectably adjustable wavelength
selector is positioned between the polychromatic light source and
the SPR optical assembly to provide selective adjustment of the
distribution of wavelengths of the excitation light directed on to
the SPR optical assembly and subsequently detected. In another
embodiment, the selectably adjustable wavelength selector is
positioned between the SPR optical assembly and the detector to
provide selective adjustment of the distribution of wavelengths
directed onto the detector and detected. The present invention also
includes embodiments having additional selectably adjustable
wavelength selectors, which may be positioned anywhere along the
optical pathway between the polychromatic optical source and the
detector. An advantage of positioning of the selectably adjustable
wavelength selector between the light source and the SPR optical
assembly is that only light having wavelengths that are detected by
the detector are exposed to the SPR optical assembly. Reducing the
intensity of light directed onto the optical assembly is beneficial
for avoiding increases in temperature of the optical assembly due
to illumination. Such temperature changes of the optical assembly
can change the refractive index of the probe region and obscure SPR
sensing measurements and images.
Selectably adjustable wavelength selectors useable in the present
invention may comprise any device or device component capable of
transmitting a selected distribution of transmitted wavelengths and
substantially preventing the transmission of other wavelengths of
light. In an exemplary embodiment of the present invention, the
selectably adjustable wavelength selector is an optical
interference filter, which is rotationally adjustable about a
rotational axis orthogonal to the plane of incidence (also
orthogonal to the incident light propagation axis or the reflected
light propagation axis). In this embodiment, rotation of the
optical interference filter selectably adjusts the distribution of
wavelengths of light that are transmitted by the optical
interference filter, particularly the center wavelength of the
distribution of transmitted wavelengths. Exemplary selectably
adjustable wavelength selectors of the present invention include,
but are not limited to, optical interference filters, etalons,
Fabry-Perot etalons, monochromators, spectrometers, prisms,
gratings and linear variable interference filters. Preferred
selectably adjustable wavelength selectors provide substantially
the same net transmittance over a range of center wavelengths
needed to measure the resonance wavelength. Preferred selectably
adjustable wavelength selectors have well characterized
transmission properties with respect to s- and p- polarized light.
In discrete wavelength operation, wavelength tuning may be used to
generate SPs that result in optimal contrast of different areas in
the probe region. In wavelength scanning operation, the center
wavelength of the distribution of transmitted wavelengths may be
continuously varied while SPR measurements or images are
collected.
Use of a selectably adjustable wavelength selector in SPR sensors
of the present invention provides the ability to tune the
wavelength distribution of excitation light, detected light or
both. The present devices and methods provide the ability to
continuously tune the wavelength distribution of excitation light,
detected light or both or a substantial range of wavelength,
preferably over a range of at least 60 nm and more preferably over
a range of several hundred nanometers. Wavelength tunability
provided by this attribute of the present invention allows changes
in SPR resonance conditions to be detected and characterized as a
function of time. Changes in SPR resonance condition may be
directly related to the refractive index of the probe region.
Therefore, wavelength tunability provided selectably adjustable
wavelength selectors of the present invention allows for accurate
quantification of physical and chemical characteristics of the
probe region. Further, wavelength tunability also provides for a
wide dynamic range of SPR sensors of the present invention.
Particularly, wavelength tunable SPR sensors and imaging devices of
the present invention may be used to detect and characterize a very
broad range of materials having different refractive indices,
thicknesses and chemical compositions. In addition, use of a
selectably adjustable wavelength selector in SPR sensors of the
present invention eliminates the need for angle modulation to
detect changes in the SPR resonance condition or determine a
resonant wavelength or distribution of resonant wavelengths.
Avoiding angle modulation SPR optical configurations is beneficial
because these configurations typically require complex rotational
optical assemblies that are spatially restrictive, costly and
sensitive to misalignment due to vibration and changes in ambient
pressure and temperature. Further, avoiding optical geometries
having complex rotational assemblies is beneficial because such
assemblies require frequent calibration and realignment.
Use of an incoherent, polychromatic light source in the present
invention has several advantages. First, use of an incoherent light
source avoids problems arising from optical interference of beam
components generated from the excitation and reflected beams.
Optical interference affects can substantially degrade SPR sensing
measurements and images due to formation of interference fringes
and speckle. In addition, incoherent light sources, such as halogen
lamps, are inexpensive, exhibit highly reproducible intensities and
are easy to optically align.
The present methods and devices are broadly applicable to any SPR
optical assembly configuration. Exemplary SPR optical assembly
assemblies useable in the present invention comprises a thin metal
film in contact with a prism and dielectric sample layer arranged
in the Kretchmann optical geometry or the Otto optical geometry.
Alternatively, sensors of the present invention may include SPR
optical assemblies comprising waveguides, fiber optic devices,
optical gratings or any combination of these components.
Any wavelength of light capable of generating SPs may be used in
the methods and devices of the present invention. Use of light
having wavelengths in the near infrared region of the
electromagnetic spectrum (about 800 nm to about 1200 nm) is
preferred for some SPR imaging applications because it provides
increased refractive index sensitivity compared to technique using
higher frequency visible light. In addition, use of the near
infrared may be beneficial for certain applications wherein the
probe region interrogated contains species that absorb in the
visible region. In preferred embodiments, the wavelengths of light
employed by SPR sensors and/or imaging devices of the present
invention are selected over the range of about 845 nm to about 857
nm.
Wavelength tunable SPR sensors of the present invention may be
operated in a variety of different operational modes. SPR
operational modes correspond to different types of SPR
measurements, different functional aspects of these devices and
different methods of using these devices. Exemplary SPR sensors of
the present invention are capable of operation in a plurality of
operating modes.
In one operational mode, SPR sensors of the present invention are
capable of measuring a distribution of resonant wavelengths
resulting SP formation. In an exemplary embodiment, the selectably
adjustable wavelength selector is adjusted to systematically vary
the wavelength distribution of detected light in a manner
generating a SP resonance curve. Preferred SP resonance curves
generated by the methods and devices of the present invention
comprise a two-dimensional plot of percent reflectivity versus the
center wavelength of the distribution of wavelengths transmitted by
the wavelength selector. Quantification of the resonant wavelength
or distribution of resonant wavelengths provides information
relating to the composition of a probe region because the resonance
condition is strongly dependent on the refractive index of the
probe region.
In another operation mode, SPR sensors of the present invention are
capable of monitoring changes in the resonant wavelength or
distribution of resonant wavelengths required for SP formation.
Monitoring changes in the distribution of resonant wavelengths is
beneficial because it provides information related to changes in
the refractive index and composition occurring in the probe region,
such as changes due to binding of chemical species to portions of
the probe region. In one embodiment, the SPR sensor is wavelength
tuned by selection of a distribution of transmitted wavelengths
resulting in formation of SPs and attenuation of reflected light.
The intensity and/or percentage reflectivity of reflected light is
monitored as a function of time over an observation interval.
Changes in the resonance condition corresponding to changes in
refractive index and chemical composition of the probe region are
observed and characterized by measuring a change in the intensity
and/or percentage reflectivity of reflected light. Alternatively,
SPR sensors of the present invention are capable of measuring
changes in the resonant wavelength or distribution of resonance
wavelengths by generating a plurality of resonance curves
corresponding to different observation intervals and/or different
experimental conditions. The measured shift in the resonant
wavelength or distribution of resonant wavelengths may be directly
related to corresponding changes in composition occurring in the
probe region. Use of a selectably adjustable wavelength selector in
these embodiments is beneficial for precisely quantifying the shift
in the resonant wavelength or distribution of resonant
wavelengths.
In another operation mode, a wavelength tunable SPR sensor of the
present invention is capable of operation as a SPR imaging device.
In this embodiment, the SPR sensor includes a two-dimensional
detector, such as a charge coupled device or two-dimensional diode
array. In a preferred embodiment, a p-polarized light beam having a
wavelength distribution capable of exciting one or more SPs is
directed at the SPR optical assembly and a first two-dimensional
distribution of reflected light intensities is measured. This first
two-dimensional distribution of reflected light intensities
comprises an image of the probe region. In some embodiments, the
distribution of reflected p-polarized light intensities must be
normalized to achieve an optimal SPR image because the combination
light source and wavelength selector of the present invention
produces wavelength dependent transmission intensities. In methods
of the present invention correcting for this effect, a s-polarized
light beam is directed at the SPR optical assembly and a second
two-dimensional distribution of reflected light intensities is
measured. Switching between p- and s- polarization orientations is
preferably achieved by adjustment of the polarizer positioned
between the polychromatic light source and the SPR optical
assembly. Comparison of first and second two-dimensional reflected
light intensity distributions generates a SPR image, which
characterizes the probe region. Preferred SPR images generated by
the present methods and devices comprise a two dimension
distribution of measured percent reflectivities. In an exemplary
embodiment, SPR images are corrected for differences in s and
p-polarization transmission properties of wavelength selectors used
in the present invention, particularly transmission properties
which vary as a function of rotational angle. Use of a selectably
adjustable wavelength selector SPR imaging devices of the present
invention is beneficial for transmitting light having distribution
of transmitted wavelengths selected to provide images having
enhanced optical quality and sharpness. Further, SPR imaging
devices and methods of the present invention are capable of
generating images exhibiting high contrast between highly
reflective regions and attenuated reflection regions.
In another aspect, the present invention provides methods of
detecting and characterizing chemical or physical interactions
between chemical species in a probe region. Particularly, a SPR
sensor of the present invention may be employed having a dielectric
sample layer operationally coupled to a reactor, such as a flow
cell or flow reactor, capable of effectively introducing chemical
species, such as atoms, molecules or ions, into the sample
dielectric layer and probe region. Exemplary reactors, are capable
of generating a flow of chemical species in a solution of other
delivery medium which contacts the probe region. The probe region
may be constructed in a manner such that it exhibits selected
chemical and/or physical properties, such as selective binding
affinities, chemical reactivities and/or physical properties. For
example, the second probe region may comprise a reactor having one
or more target chemical species, such as biological polymers,
immobilized on the reactor surface. In an exemplary embodiment, the
sensing surface of the thin conducting layer is chemically modified
to provide selective affinity, reactivity, bonding or other
chemical and/or physical properties. Deposition of selected
materials directly onto the surface of the conducting layer, such
as carboxymethylated dextran, may facilitate covalent attachment of
biopolymers such as proteins or oligonucleotides to sensing
surfaces of the present invention. In these embodiments,
introduction of one or more interacting species to the reactor may
result in binding, chemical reaction or physical interaction
between target and interacting species, thereby changing the
refractive index in the probe region. Use of SPR sensors of the
present invention may be used to detect changes in the refractive
index of probe and, thereby characterize the nature of chemical or
physical interaction of target chemical species and an interacting
species.
In an exemplary embodiment, the SPR sensor generates at least one
reference measurement corresponding to the refractive index of the
probe region prior to introduction of interacting species.
Interacting species are introduced into the reactor and permitted
to interact with the target species in the probe region. The SPR
sensor generates at least one analytical measurement, which is
compared to the reference measurement to detect a change in the
refractive index of said probe region. In an exemplary embodiment,
analytical measurements are repeatedly acquired and compared to
each other to characterize changes in refractive index as a
function of time. Such changes may be related to the chemical and
physical nature of the interaction between interacting species and
target species. Exemplary methods of the present invention are
capable of determining binding affinities, rate constants,
equilibrium constants and thermodynamic parameters that
characterize the interaction between target species and interacting
species.
The SPR sensing and imaging methods and devices of the present
invention are broadly applicable for detecting and characterizing
virtually any material capable of changing the refractive index. In
particular, the present methods are particularly useful for
detecting chemical species including, but not limited to,
biological polymers, such as proteins, peptides, oligonucelotides,
glycoproteins, DNA, RNA, polysaccharides, and lipids and aggregates
thereof. An advantage of the present methods and devices is that
they provide sensitive detection methods which do not require
pre-detection chemical labeling processes which are time consuming,
costly and may substantially affect the chemical and/or physical
properties of the labeled chemical species. Other advantages of the
present sensing and imaging methods is that they provide very high
time resolution, high sensitivity up to about 100 fM and require
very low sample volumes.
The present invention provides methods and devices broadly
applicable to any measurement technique or other processes which
involves the formation of SPs. Particularly, wavelength tunability
of the devices of the present invention provides efficient SP
excitation. For example, wavelength tunable SP devices of the
present invention may be used for effective excitation of
photoluminescent materials in the SPR probe region. In an exemplary
embodiment, devices of the present invention are used to generate
SPs capable of exciting fluorescent or phosphorescent transitions
of chemical species in the probe region, particularly in chemical
species bound to the probe region. These devices may include a
second detector positioned in optical communication with such
fluorescent materials, which is capable of quantifying the
intensity of SP induced fluorescence. Alternatively, wavelength
tunable SP devices of the present invention may provide a means of
delivering energy to materials in a reaction region to induce
chemical or physical changes in the material.
In another aspect, the present invention provides a method of
sensing the refractive index of a probe region comprising the steps
of: (i) passing light from a polychromatic light source through a
polarizer, thereby generating light propagating along an incident
light propagation axis; (ii) directing said light onto an optical
assembly comprising a dielectric layer, a sample dielectric layer
and a conducting layer positioned between the dielectric layer and
the dielectric sample layer, thereby generating light propagating
along a reflected light propagation axis, wherein a portion of said
dielectric sample layer adjacent to said conducting layer comprises
the probe region; (iii) passing said light through a selectably
adjustable wavelength selector positioned in the optical path
between said light source and a detector; (iv) detecting said light
with said detector, thereby sensing the refractive index of the
probe region, and (v) adjusting said selectably adjustable
wavelength selector to transmit light having a distribution of
wavelengths selected to generate surface plasmons on a surface of
said conducting layer in contact with said dielectric sample
layer.
In another aspect, the present invention provides a method of
generating an image of a probe region comprising the steps of: (i)
passing light from a polychromatic light source through a
polarizer, thereby generating light propagating along an incident
light propagation axis; (ii) directing said light onto an optical
assembly comprising a dielectric layer, a sample dielectric layer
and a conducting layer positioned between a dielectric layer and
dielectric sample layer, thereby generating light propagating along
a reflected light propagation axis, wherein a portion of said
dielectric sample layer adjacent to said conducting layer comprises
the probe region; (iii) passing said light through a selectably
adjustable wavelength selector positioned in the optical path
between said light source and a detector; (iv)detecting said light
with said detector, thereby generating said image of said probe
region, and (vi) adjusting said selectably adjustable wavelength
selector to transmit light having a distribution of wavelengths
selected to generate surface plasmons on a surface of said
conducting layer in contact with said dielectric sample layer.
In another aspect, the present invention provides a method of
detecting a change in the refractive index of a probe region
comprising the steps of: (i) passing light from a polychromatic
light source through a polarizer, thereby generating light
propagating along an incident light propagation axis; (ii)
directing said light onto an optical assembly comprising a
dielectric layer, a sample dielectric layer and a conducting layer
positioned between a dielectric layer and dielectric sample layer,
thereby generating light propagating along a reflected light
propagation axis, wherein a portion of said dielectric sample layer
adjacent to said conducting layer comprises the probe region; (iii)
passing said light through a selectably adjustable wavelength
selector positioned in the optical path between said light source
and a detector, wherein said selectably adjustable wavelength
selector is adjusted to transmit incident light having a
distribution of wavelengths selected to generate surface plasmons
on a surface of said conducting layer in contact with said
dielectric sample layer; (iv) detecting said light with said
detector, thereby generating at least one reference measurement,
(v) detecting said light with said detector, thereby generating at
least one analytical measurement, and (vi) comparing said reference
measurement and said analytical measurement to detect said change
in the refractive index of said probe region.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic drawing showing a side plan view of a SPR
imaging device of the present invention having a selectably
adjustable wavelength selector positioned between the polychromatic
light source and the SPR optical assembly.
FIG. 2 is a schematic drawing showing a side plan view of a SPR
imaging device of the present invention having a selectably
adjustable wavelength selector positioned between the SPR optical
assembly and the detector.
FIG. 3 is a schematic drawing showing a side plan view of an
exemplary selectably adjustable wavelength selector comprising an
optical interference filter. As illustrated in FIG. 3, the optical
interference filter is selectably rotatable about a rotation axis
which is orthogonal to the plane of incidence.
FIG. 4 is a plot of center wavelength as a function of tilt angle
for two interference filters having center wavelengths at normal
incidence of about 850 nm (filled diamonds) and about 880 nm (open
diamonds).
FIG. 5 is a schematic diagram illustrating a top plane view of an
exemplary SPR imaging device based on the Kretschmann SPR
configuration.
FIG. 6 is a correction curve for correcting acquired SPR images for
polarization dependent transmission of light through an optical
interference filter.
FIG. 7 is the expected response of an exemplary SPR imaging device
for changes in sample refractive index.
FIG. 8 shows the optimal center imaging wavelength as a function of
change in refractive index from a base refractive index of
water.
FIG. 9 shows a series of normalized images of sucrose solutions
having a range of various refractive indexes measured by an
exemplary sensor of the present invention.
FIG. 10 shows a plot of the experimental response of an exemplary
SPR sensor. As shown, the response of the system is linear for
changes in refractive indices less than 3.times.10.sup.-3.
FIG. 11 shows the signal-to-noise ratio as a function of number of
pixels averaged for both uncorrected (A, bottom plot) and corrected
(B, top plot) SPR data.
FIG. 12 shows a series of images taken of a thiol and water
patterns with an optical interference filter positioned a several
different tilt angles. FIG. 12A corresponds to a center wavelength
of 857 nm, FIG. 12B corresponds to a center wavelength of 852 nm,
FIG. 12C corresponds to a center wavelength of 845 nm, FIG. 12D
corresponds to a center wavelength of 830 nm and FIG. 12E
corresponds to a center wavelength of 814 nm.
FIGS. 13A and B shows images of thiol patterns with minimum feature
sizes of approximately 100 .mu.m (FIG. 13A, left side) and
approximately 50 .mu.m (FIG. 13B, right side) generated by an
exemplary SPR senor of the present invention.
FIGS. 14A-D show images generated upon the adsorption of protein
bovine serum albumin (BSA) onto a gold surface. The image in FIG.
14A shows a small region of the reactor with a background of water.
FIG. 14B shows an image of the same region of the reactor with a 2
mg ml.sup.-1 solution of BSA in phosphate buffered saline (PBS).
FIG. 14C shows an image of the same region with a background of
water after pumping water through the reactor to remove all unbound
protein. FIG. 14D shows a difference image resulting from
subtraction of images in FIG. 14A and FIG. 14C.
DETAILED DESCRIPTION OF THE INVENTION
Referring to the drawings, like numerals indicate like elements and
the same number appearing in more than one drawing refers to the
same element. In addition, hereinafter, the following definitions
apply:
"Chemical species" refers generally and broadly to a collection of
one or more atoms, molecules and/or macromolecules whether neutral
or ionized. In particular, reference to chemical species in the
present invention includes but is not limited to biopolymers.
Chemical species in a liquid sample may be present in a variety of
forms including acidic, basic, molecular, ionic, complexed and
solvated forms. Chemical species also includes non-covalently bound
aggregates of molecules. Chemical species includes biological
molecules, i.e. molecules from biological sources, including
biological polymers, any or all of which may be in the forms listed
above or present as aggregates of two or more molecules.
"Distribution of transmitted wavelengths" refers to a
two-dimensional distribution of the intensities of light of
different wavelengths transmitted by a wavelength selector, such as
an optical interference filter, monochromator or spectrometer. A
distribution of transmitted wavelengths may be characterized in
terms of center wavelength, bandwidth and intensity profile of
transmitted wavelengths. In the present invention, the distribution
of transmitted wavelengths of light detected by a detector is
determined by the combination of the optical properties of the
light source and the selectably adjustable wavelength selector. In
an exemplary embodiment, the distribution of transmitted
wavelengths of light directed on an SPR optical assembly and/or
detected by a detector has a substantially Gaussian intensity
profile and center wavelength corresponding to the wavelength of
light having the largest intensity. Wavelength tunable SPR sensors
and imaging devices of the present invention provide excitation
light and/or detected light having a selectably adjustable
distribution of transmitted wavelengths.
"Center wavelength" is a characteristic of a distribution of
transmitted wavelengths of light. In some embodiments, center
wavelength refers to the midpoint wavelength in a distribution of
transmitted wavelengths. In other embodiments, the center frequency
refers to the transmitted wavelength in a distribution of
wavelength having the largest intensity. In other embodiments,
center wavelength refers to the average wavelength in a
distribution of transmitted wavelengths. The center wavelength
typically corresponds to the wavelength in a distribution of
transmitted wavelength having the largest intensity for wavelength
distributions having Gaussian or Lorentizian intensity profile.
"Light source" refers to any device or material capable of
generating electromagnetic radiation or a plurality of devices or
materials capable of generating electromagnetic radiation.
Preferred light sources in the present invention are capable of
generating light in the near infrared region of the electromagnetic
spectrum (about 800 nm to about 1200 nm). In an exemplary
embodiment useful for avoiding optical interference affects in SPR
imaging and sensing applications, a light source of the present
invention generates incoherent light. Light sources useable in the
methods and devices of the present invention include halogen lamps,
light emitting diodes, fluorescent lamps, tungsten-filament lamps,
grey body light sources and black body light sources.
"Bandwidth" refers to a characteristic of a distribution of
transmitted wavelengths of light. Bandwidth may be defined in terms
of the full width at half maximum of an intensity profile of a
distribution of transmitted wavelengths, which refers to the full
width at an transmittance equal to one half of the maximum
transmittance. In exemplary embodiments of the present invention
bandwidth of detected light is primarily determined by the
transmission properties of a selectably adjustable wavelength
selector, such as optical interference filter. The transmission
bands of exemplary selectably adjustable wavelength selectors of
the present invention are selected over the range of about 1 nm to
about 100 nm and more preferably 1 to about 20 nm in some
embodiments. Use of wavelength selectors capable of providing a
distribution of transmitted wavelengths characterized by a large
bandwidth (>10 nm) is useful in some embodiments for increasing
signal-to-noise ratio.
"Conducting layer" refers to a layer comprising a conductor
material, such as a metal, or a semiconductor material. Conducting
layers support the formation of surface plasmons and are used as
sensing surfaces in the present invention. Preferred conducting
layers in the present invention are thin (<50 nm) gold or silver
layers.
"Dielectric sample layer" refers to a dielectric layer positioned
adjacent to the surface of a conducting layer having surface
plasmons thereon. Dielectric sample layers of the present invention
include probe regions close to the sensing surface of a conducting
layer. Probe regions of the present invention comprise a volume
adjacent to the surface of a conducting layer having surface
plasmons thereon (a sensing surface) having a depth that is defined
by the decay length of the surface plasmons into the dielectric
sample layer. SPR sensors and imaging devices of the present
invention are capable of sensing, monitoring and characterizing
changes in the refractive index of a probe region. Dielectric
sample layers and probe regions may be operational connected to
flow cells and/or flow reactors for introduction of material to the
dielectric sample layer and/or probe region. In these embodiments,
selection of the flow conditions of the flow cell or flow reactor
may adjust the composition of the dielectric sample layer and the
probe region. Alternatively, dielectric sample layers and probe
regions may be operational connected to static cells and/or static
reactors.
"Selectably adjustable wavelength selector" refers to a device,
device component or combination of optical components capable of
selecting the distribution of wavelengths of light which are
transmitted through the wavelength selector. "Selectably adjustable
wavelength selector" also refers to a device, device component or
combination of optical components capable of selecting the
distribution of wavelengths of light which are substantially
prevented from transmitting through the wavelength selector.
Selectably adjustable wavelength selectors of the present invention
may transmit a distribution of transmitted wavelengths
characterized by a center wavelength, bandwidth and intensity
profile. Exemplary selectably adjustable wavelength selectors of
the present invention include, but are not limited to, optical
interference filters, etalons, Fabry-Perot etalons, fiber optic
interferometric filters, fiber optic devices, fiber Fabry-Perot
filters, monochromators, spectrometers, gratings and/or prisms,
slits or any combinations thereof. Exemplary optical interference
filters of the present invention are capable of selectably
adjusting the distribution of transmitted wavelengths by rotation
about a rotational axis which is oriented orthogonal to an incident
or reflected beam axis.
"Surface plasmon resonance sensor" or "SPR sensor" are used
synonymously and refer to any device or device component capable of
monitoring, detecting or characterizing changes in the refractive
index of a probe region using excitation of surface plasmons. In an
exemplary embodiment, SPR sensors detect changes in the refractive
index of a probe region located proximate to a sensing surface
having surface plasmons localized thereon. Exemplary SPR sensors
comprise SPR imaging devices which are capable of generating an
image of a probe region corresponding to refractive indices and/or
composition of the probe region. Alternatively, SPR sensors of the
present invention generate surface plasmons capable of exciting
photoluminescent materials positioned proximate to one or more
surfaces of a conducting layer.
"SPR optical assembly" refers to any combination of optical
components which are capable of coupling radiant energy into
surface plasmons. In an exemplary embodiment, a SPR optical
assembly of the present invention comprises a dielectric layer, a
dielectric sample layer and conducting layer arrange in the
Kretschmann optical configuration or Otto optical configuration.
Alternatively, SPR optical assemblies may comprise waveguides,
fiber optic devices or diffraction gratings. SPR optical assemblies
of the present invention may include a number of optical components
including, but not limited to, prisms, thin gold films, thin silver
films, thin semiconductor films, flow reactors, static reactors,
microfluidic devices, fluid channels, optical alignment systems,
rotational stages or any combination of these components.
"Tilt angle" is a characteristic of rotational position. In
exemplary embodiments, tilt angle refers to rotational orientations
of an optical interference filter relative to normal incidence with
respect to the incident light propagation axis or reflected light
propagation axis. Specifically, tilt angle refers to the angular
deviation of the surface of a rotated optical component, such as an
optical interference filter, as measured relative to the incident
light propagation axis or reflected light propagation axis.
Exemplary surfaces of optical interference filters of the present
invention may be oriented at tilt angles ranging from 0.degree. to
about 60.degree., more preferably from 0.degree. to about
35.degree.
In the following description, numerous specific details of the
devices, device components and methods of the present invention are
set forth in order to provide a thorough explanation of the precise
nature of the invention. It will be apparent, however, to those of
skill in the art that the invention can be practiced without these
specific details.
This invention provides methods, devices and device components for
sensing changes in the refractive index and composition of a probe
region proximate to a sensing surface. In particular, wavelength
tunable SPR sensing devices and images devices are provided which
are capable of detecting SPR conditions and generating SP resonance
curves at a constant angle of incidence. Further, the present
invention provides methods and devices of generating SPR images of
a probe region in a sample dielectric layer.
FIG. 1 schematically illustrates a side plan view of a SPR imaging
device of the present invention having a selectably adjustable
wavelength selector positioned between a polychromatic light source
and a SPR optical assembly. The exemplary SPR imaging device 100
comprises a polychromatic light source 110 in optical communication
with polarizer 130, optical interference filter 140, SPR optical
assembly 150 and two-dimensional detector 155. As shown in FIG. 1,
exemplary SPR imaging device 100 may optionally include light
collimation element 170 comprising lenses 175 and pin hole 177
positioned between light source 110 and polarizer 130. Further, SPR
imaging device may optional comprise collecting and imaging optical
element 158 positioned between optical assembly 150 and detector
155.
Incident light 160 from optical source 110 is collimated by
collimation element 170 and propagates along incident light
propagation axis 180. Incident light 160 is passed through
polarizer 130 positioned to intersect light propagation axis 180,
which is capable of selecting the polarization state of incident
light 160. Polarizer 130 is preferably capable of selected
substantially p-polarized or s-polarized orientations of incident
light 160 and also of rapidly switching between selected
p-polarization and s-polarization orientations. A selected
distribution of transmitted wavelengths of polarized incident light
160 passes through optical interference filter 140, which is
positioned to intersect light propagation axis 180. In the
embodiment illustrated in FIG. 1, optical interference filter 140
is selectably rotatable about a rotational axis which is oriented
orthogonal to the incident light propagation axis 180 (due to the
perspective of FIG. 1, the rotational axis of optical interference
filter 140 is not shown but is oriented such that it comes out of
the plane of the drawing). Two different rotational orientations of
optical interference filter 140 are shown in FIG. 1. In this
embodiment, the wavelength distribution of transmitted light may be
selectably adjusted by rotation of optical interference filter 140.
In a preferred embodiment, optical interference filter 140 is
mounted on a rotation stage (not shown in FIG. 1) so that the angle
of the filter face with respect to the incident light propagation
axis may be selectively varied, thus, varying the wavelengths of
light that are passed by the filter. Therefore, the rotational
position of optical interference filter 140 determines the
wavelength distribution of light which is directed to optical
assembly 150 and subsequently detected by detector 155
Light having a selected wavelength 200 is directed onto SPR optical
assembly 150, which comprises a prism 210, a thin conducting layer
220, and a dielectric sample layer 230 arranged in the Kretschmann
optical configuration. A preferred thin conducting layer 220 of the
present invention are gold or silver layers having a thickness
ranging from about 30 nm to about 60 nm. A preferred dielectric
sample layer 230 comprises a probe region 270 operationally coupled
to a reactor or cell, such as a flow cell, static cell, flow cell
reactor or static cell reactor, capable of introducing material
into a probe region 270 proximate to the surface of thin conducting
layer 220. Illumination of the SPR optical assembly 150 at angles
of incidence resulting in total internal reflection generates light
240 propagating along a reflected light propagation axis 250 that
is detected by two-dimensional detector 155. Optionally, light from
optical assembly 150 may be collected by optical collection and
focusing element 158 prior to detection to enhance detection
sensitivity and resolution.
At least a portion of light having wavelengths that satisfy the SPR
resonance condition is not reflected by optical assembly 150.
Rather, this radiant energy is converted into SPs on sensing
surface 260, which comprises the surface of conducting layer 220 in
contact with dielectric sample layer 230. The resonance condition
controlling conversion of radiant energy to surface plasmons is
strongly dependent on the refractive index of a probe region 270
proximate to the sensing surface 260. Detection of light reflected
by optical assembly 150 to detector 155 is capable of
characterizing which wavelengths of light are converted to SPs and
the extent of this process. As shown in FIG. 1, preferred optical
geometries of SPR sensors and imaging devices of the present
invention have a constant angle of incidence selected to generate
total internal reflection of the incident beam upon illumination of
the SPR optical assembly. The present invention also includes,
however, embodiments wherein the angle of incidence is selectably
adjustable. These embodiments correspond to SPR sensors and imaging
devices that are both angle and wavelength tunable.
FIG. 2 shows an exemplary SPR imaging device 300 having an
alternative optical configuration. In this optical configuration,
optical interference filter 140 is positioned to intersect
reflected light optical propagation axis 250. Similar to the
optical configuration shown in FIG. 1, optical interference filter
is selectably rotatable about a rotational axis. In exemplary SPR
imaging device 300, optical interference filter 140 is selectably
rotatable about a rotational axis that is oriented orthogonal to
the reflected light propagation axis 250 (due to the perspective of
FIG. 2, the rotational axis of optical interference filter 140 is
not shown but is oriented such that it comes out of the plane of
the drawing). In this embodiment, the wavelength distribution of
transmitted light may be selectably adjusted by rotation of optical
interference filter 140. Only one rotational orientation of optical
interference filter 140 is shown in FIG. 2. In an exemplary
embodiment, optical interference filter 140 is mounted on a
rotation stage (not shown in FIG. 2) so that the angle of the
filter face with respect to the reflected light propagation axis
may be selectively varied, thus, varying the wavelengths of light
that are passed by the filter. Therefore, the rotational position
of optical interference filter 140 determines the wavelength
distribution of light detected by detector 155. As shown in FIG. 2,
SPR imaging device 300 may optionally include cutoff filter 302
positioned between light source 110 and optical assembly 150. In an
exemplary embodiment cutoff filter 302 is a 700 nm long pass filter
and/or a 1000 nm short pass filter, which reduces the intensity of
light having wavelengths less than 700 nm to minimize heating of
optical assembly 150 by incident light.
To generate a SPR image, polarizer 130 is adjusted to transmit
p-polarized light and optical interference filter 140 is adjusted
to transmit light having a distribution of wavelengths satisfying
the SP resonance condition for a particular probe region
composition and refractive index. Detector 155 detects light
reflected from optical assembly 150, thereby generating a first
two-dimensional distribution of reflected light intensities
corresponding to p-polarized light. In some embodiments, the
two-dimensional distribution of reflected p-polarized light
provides a SPR image of the probe region. Use of a combination of
light source and wavelength selector having intensities which vary
with wavelength often requires normalization of the measured
p-polarized reflected light intensities in order to calculate an
image in terms of percent reflectivity. To convert the reflected
intensities corresponding to p-polarized light into percent
reflectivities, polarizer 130 is adjusted to transmit s-polarized
light and a second two-dimensional distribution of reflected light
intensities is generated corresponding to s-polarized light. An
image of the probe region in terms of percent reflectivity is
generated by taking the ratio of p-polarized intensity to
s-polarized intensity at each pixel location.
At larger filter rotation angles, the polarization-dependent
transmission effects of the interference filter become significant.
Specifically, the intensity of transmitted s-polarized light
decreases and the center wavelength is shifted to shorter
wavelengths as compared to the transmitted p-polarized light
through the same interference filter. As a result, at larger filter
rotation angles, normalized images must include a correction factor
for this effect. The correction factor for any imaging angle can be
simply determined by measuring the intensities of p-polarized and
s-polarized light passed by the filter in the absence of surface
plasmon generation. The ratio (intensity p-polarized)/(intensity
s-polarized) itself can be used to correct for
polarization-dependent transmission effects of the interference
filter. For example, each measured percent reflectivity value may
be divided by the ratio of the intensity of p-polarized light to
the intensity of s-polarized light corresponding to the center
wavelength of the transmitted light distribution to correct for
polarization dependent transmission affects. In one embodiment,
(the intensity p-polarized)/(intensity s-polarized) correction
factor is measured at several different imaging angles and the data
is fit by a third order polynomial function to generate a
correction curve for the system. The correction curve is then used
to obtain the correction factor for any distribution of transmitted
wavelengths.
FIG. 3 is an expanded view of optical interference filter 140
showing a plurality of rotational orientations relative to normal
incidence with respect to incident light propagation axis 180 or
reflected light propagation axis 250. Specifically, rotation
orientations corresponding to a first tilt angle 350 and a second
tilt angle 360 are shown. First tilt angle 350 is smaller than
second tilt angle 360. As shown in FIG. 3, in the context of some
embodiments of the present invention, tilt angle refers to angular
deviation as measured relative to an angular orientation of optical
interference filter such that it is orthogonal to the incident
light propagation axis or reflected light propagation axis.
Alternatively expressed, tilt angle is 90 degrees minus the angle
between the normal to the plane defined by the filter face and the
incident beam axis. In an exemplary embodiment, the optical
interference filter transmits light having a distribution of
wavelengths that is characterized by a center wavelength, bandwidth
and wavelength intensity profile. Preferred bandwidths range form
about 1 nm to about 30 nm and preferred wavelength intensities
profiles are substantially Gaussian shaped or Lorentzian shaped.
Exemplary optical interference filters provide center frequencies
which are tunable over a range of about 60 nm and more preferably
about 100 nm.
In one embodiment, rotation of optical filter 140 shifts the center
wavelength of the distribution of transmitted wavelengths to
shorter wavelengths. In an exemplary embodiment wherein the optical
interference filter comprises a Fabry-Perot etalon, the center
wavelength of the optical interference filter is provided by the
expression:
.lamda..function..theta..lamda..function..times..times..theta.
##EQU00005## wherein .lamda..sub.center is the center wavelength of
the distribution of transmitted wavelengths, .theta..sub.tilt is
the tilt angle, .lamda..sub.center(0) is the center wavelength at
normal incidence with respect to the reflected or incident light
propagation axes and n is the refractive index of the optical
interference filter. For optical interference filters comprising
Fabry-Perot etalons n is the half wavelength thick layer of the
filter.
An exemplary optical interference filter useable in SPR sensors and
imaging devices of the present invention has a full width at half
maximum bandwidth of about 10 nm at a normal incidence (angle
between the normal to the filter face and the incident light axis).
FIG. 4 show a plot of center wavelength as a function of tilt angle
for two interference filters having center wavelengths at normal
incidence of about 850 nm (filled diamonds) and about 880 nm (open
diamonds). As shown in FIG. 4, the center wavelengths passed by the
filters shift by about 65 nm for a variation in tilt angle from 0
to 35.degree.. The variation of the center wavelength agreed with
the values predicted using Equation VI. The intensity distribution
of wavelengths remained substantially Gaussian up to tilt angles of
about 35.degree.. The width of the Gaussian intensity profiles,
however, increases by approximately 4% as the filter is titled from
0.degree. to about 20.degree.. Above a tilt angle of about
20.degree., the width of the intensity distribution increases more
rapidly with angle, up to an additional 20%.
The range in wavelength tuning needed to optimally image samples
that vary in refractive index from about 5.times.10.sup.-5 to about
3.times.10.sup.-3 from a baseline of water on bare Au (refractive
index equal to 1.328 at .about.850 nm) was estimated using a
3-layer SPR model. The results of these calculations indicate that
the optimal range of wavelengths is from about 845 nm to about 857
nm for characterizing the expected change in SP resonant
wavelength. This range spans less than 15 nm and, thus, is easily
covered by the wavelength shift range provide by a single optical
interference filter.
SPR sensors and imaging devices of the present invention may
comprise stand-alone instruments. Alternatively, the SPR sensors
and imaging devices of the present invention may be integrated into
other devices or used as device components in instruments. The
sensors of the present invention may be coupled to reactors, flow
cells, static cells, flow cell reactors, static reactors,
microfluidic devices, biological system analyzers, instruments for
characterizing the interactions between molecules, and drug
screening instruments. Flow cells operationally coupled to the
sensors and imaging devices of the present invention are useful for
delivering chemical species to the probe region. For example, the
SPR sensors of the present invention may be combined with a
microfluidic fluid delivery device to introduce materials into the
probe region. In an exemplary embodiment, the sensing surface of a
sensor of the present invention comprises one wall of a
microfluidic flow cell. SPR sensing measurements may be conducted
during conditions of continuous liquid flow over the surface or
static flow conditions. Use of a microfluidic flow system is
beneficial because it provides precise control over the time-point
and duration of sample delivery to the probe region.
All references cited in this application are hereby incorporated in
their entireties by reference herein to the extent that they are
not inconsistent with the disclosure in this application. It will
be apparent to one of ordinary skill in the art that methods,
devices, device elements, materials, procedures and techniques
other than those specifically described herein can be applied to
the practice of the invention as broadly disclosed herein without
resort to undue experimentation. All art-known functional
equivalents of methods, devices, device elements, materials,
procedures and techniques specifically described herein are
intended to be encompassed by this invention.
EXAMPLE 1
Characterization of an Exemplary SPR Sensor
The ability of SPR sensors of the present invention to sense
changes in the refractive index of a probe region was verified by
experimental and computational studies. Specifically, it is a goal
of the present invention to provide SPR sensors capable of
sensitively detection and characterization changes in the
refractive index of a probe region. Further, it is a goal of the
present invention to SPR sensors providing a large dynamic range,
which are capable of probing materials having a wide range of
refractive indices.
To achieve the aforementioned goals, detection sensitivities and
dynamic ranges of an exemplary SPR sensor were computationally
modeled and evaluated by monitoring the refractive indices of low
concentration sucrose solutions. The exemplary SPR sensor 500
employed in these studies is based on the Kretschmann configuration
and is shown in FIG. 5. The polychromatic light source is a 150 W
quartz halogen lamp 510 (Dolan-Jenner, Lawrence, Mass.) coupled to
a multi-fiber light pipe 515 (Edmund Industrial Optics, Barrington,
N.J.). Light from the source passes through iris 520 and is
collected by an achromatic lens 525 (Edmund Industrial Optics,
Barrington, N.J.) and focused at a pinhole 530 (100 .mu.m in
diameter, Edmund Industrial Optics, Barrington, N.J.). A second
achromatic lens 535 (Edmund Industrial Optics, Barrington, N.J.)
collects light from the pinhole 525 and forms a collimated beam.
This expanded and collimated beam passes through a polarizer 540
(Edmund Industrial Optics, Barrington, N.J.). The polarizer is
mounted onto a motorized rotation stage 545 (Newport Corporation,
Irvine, Calif.) so p-polarized and s-polarized images can be
acquired conveniently. The light then passes through an
interference filter 550 (Edmund Industrial Optics, Barrington,
N.J.) that selects a narrow band (10 nm FWHM) of operating
wavelengths in the near infrared to optimally contrast the range of
refractive indexes in the sample. The filter is mounted onto a
motorized rotation stage 555 (Newport Corporation, Irvine, Calif.)
so that the angle of the filter face with respect to the collimated
source beam may be varied, thus varying the wavelengths of light
that are passed by the filter. Rotation of the filter over tilt
angles of about 35.degree. form normal incidence, results in
variation of the wavelengths passed by the filter by .about.70 nm
toward shorter wavelengths.
The SPR optical assembly 560 comprises a prism, thin gold film and
a flow reactor. The entrance and exit surfaces 565 and 570 of the
prism were custom-ground (Matthew's Optical, Poulsbo, Wash.) to be
perpendicular to the source beam for an incident angle of
64.8.degree. at the metal surface. Light reflected form the SPR
optical assembly passes through an imaging lens 575 (Edmund
Industrial Optics, Barrington, N.J.) to form a focused image
(magnification<1) at the CCD detector 580 (Retiga EX, Qlmaging,
Burnaby, Canada). The area of sample interrogation is circular and
.about.16 mm in diameter. Data acquisition is performed with
software written in-house using Labview 6.1 (National Instruments,
Austin, Tex.).
Use of a light source and interference filter combination providing
incident light intensities that vary with center wavelength
requires normalization of the p-polarized signal by the s-polarized
signal. Further, polarization-dependent transmission effects of the
interference filter become significant at larger filter rotation
angles. As compared to transmitted p-polarized light, the intensity
of transmitted s-polarized light decreases and the center
wavelength is shifted to shorter wavelengths as tilt angle is
increased. As a result, at larger filter rotation angles,
normalized images must include a correction factor for this effect.
Correction factors were determined by measuring the intensities of
p-polarized and s-polarized light passed by the optical
interference filter in the absence of surface plasmon formation.
The ratio (intensity p-polarized)/(intensity s-polarized) itself
was used to correct for polarization-dependent transmission effects
of the interference filter. Each measured percent reflectivity
value was divided by the correction factor to correct for
polarization dependent transmission affects.
FIG. 6 is a correction curve for correcting acquired SPR images for
polarization dependent transmission of light through the
interference filter. The diamond data points in FIG. 6 show the sum
of the intensity of p-polarized light divided by the sum of the
intensity of s-polarized light for ten different filter rotation
angles in the absence of surface plasmons. To estimate the
variability in the correction procedure, data was taken from five
different regions of the source beam. Each point in the plot shown
in FIG. 6 is the average of 400 pixels. The error in the data
points increases with increasing filter tilt angle, from .ltoreq.1%
for rotation angles less than 24.degree. to 6% at an angle of
36.degree.. The variation between different runs is considerably
smaller, .ltoreq.0.6% for all filter tilt angles. Also, shown in
FIG. 6 is the correction curve, a 3.sup.rd order polynomial,
obtained from the data. As is apparent from the plot, the
correction factor at rotation angles of less than 25.degree. is
small (less than 1.3) but increases rapidly for the larger rotation
angles, up to 2.3 at 36.degree..
Using a 3-layer Fresnel model, the predicted response of an
exemplary SPR sensor was calculated for a probe region refractive
index that spans 4 orders of magnitude, 1.times.10.sup.-6 to
1.times.10.sup.-2. The calculations assumed a base refractive index
of water equal to 1.328 at .about.850 nm. The expected response of
the SPR sensor for changes in sample refractive index is shown in
FIG. 7. The data shown in FIG. 7 takes into account the
experimentally measured transmission band of our filter equal to
about 10 nm. Each diamond data point corresponds to the expected
response of the instrument for a given change in sample refractive
index at the optimal center imaging wavelength for that sample
change in refractive index. The square data points correspond to
the expected response of the instrument at a single center
wavelength setting of 853 nm. The SPR sensor is expected to have a
linear response up to a change in refractive index of
.about.3.times.10.sup.-3 (for comparison, the adsorption of a
monolayer of the protein bovine serum albumin onto the Au surface
corresponds to a refractive index change of
.about.1.times.10.sup.-3). Also of note is the effect on the sensor
response when acquiring data at the single center wavelength of 853
nm. FIG. 8 shows the optimal center imaging wavelength as a
function of change in refractive index from a base refractive index
of water. An imaging wavelength of 853 nm is only optimal for a
change in refractive index of .about.3.times.10.sup.-3. However,
for changes in refractive index of <3.times.10.sup.-3 the
response measured at a center wavelength of 853 nm is near that
expected at the optimal center imaging wavelength. For larger
changes in refractive index, the response measured at a center
imaging wavelength of 853 nm is significantly less than that
expected at the optimal center imaging wavelength, up to .about.10%
decrease for a change in sample refractive index of 0.01.
The experimental response of the exemplary SPR sensor was
investigated using a series of low concentration sucrose
(Sigma-Aldrich Inc., St. Louis, Mo.) solutions. The refractive
index of each sucrose solution was measured with a refractometer
(Milton Roy Company, Ivyland, Pa.). The system uses standard size
soda lime glass microscope slides (Fisher Scientific, Hampton,
N.H.), cleaned in Nanostrip.TM. solution, and then deposited with 1
nm Cr and 450 nm Au. Before use on the imaging system, the Au
coated slides were cleaned in a 1:1:5 solution of 30% hydrogen
peroxide, ammonium hydroxide, and ddI water. The slides were then
placed in a 0.2 mM ethyleneglycol-terminated thiol solution for
24-72 hours in a darkened, nitrogen atmosphere to allow for the
formation of a non-fouling self-assembled monolayer. FIG. 9 shows a
series of normalized images of solutions of various refractive
indexes. All images were taken with a center imaging wavelength of
.about.850 nm. The images A through D show the same region of the
flow reactor with solutions of refractive index 1.3338, 1.3343,
1.3346, and 1.3354, respectively. After the introduction of each
sucrose solution, the reactor and system was flushed with ddI
water. Analysis of the signal in the region after each ddI water
rinse indicates little nonspecific adsorption, .ltoreq.7% variation
in the signal. FIG. 10 shows a plot of the experimental response of
an exemplary SPR sensor. As shown, the response of the system is
linear for changes in refractive indexes <3.times.10.sup.-3.
The detection limit of the exemplary SPR sensor was investigated
using a sample of ddI water. Images were taken at 2 sec intervals
and an exposure time of 1.2 s over a time period of .about.3
minutes. The p-polarized images were normalized with an s-polarized
image to obtain percent reflectivity using an s-polarized image and
data averaged from an area of 100 pixels. The water sample showed a
50% reflectivity with a standard deviation of 0.13%. Thus, the
detection limit of the instrument is .about.4 times this standard
deviation, or 0.5%. This reflectivity corresponds to a lower limit
in the detectable change in refractive index of
.about.5.times.10.sup.-5.
No further increases in the signal to noise ratio (SNR) were
obtained by averaging over >100 pixels unless the signal was
also normalized for temporal changes in the SPR sensor. With the
appropriate reference normalization, however, the SNR increases, as
expected, with the square of the signal intensity. FIG. 11 shows
the SNR ratio as a function of number of pixels averaged for both
uncorrected (A, bottom plot) and corrected (B, top plot) data SPR
data. The data consisted of a series of 100 images (800 ms exposure
time) of the source beam taken at 2 s intervals. Specifically, a
factor of 10.sup.2 increase in the intensity of our signal would
yield a factor of 10 increase in the SNR or a detection limit of
.about.5.times.10.sup.-6. Additionally, the SNR was increased
further by hardware modifications to the system that result in an
increase in the source intensity.
EXAMPLE 2
SPR Images of Thiol Patterns and Protein Bovine Serum Albumin on
Gold Surfaces
To assess the sensitivity and spatial resolution of SPR imaging
devices of the present invention, SPR images of thiol patterns were
generated by an exemplary SPR sensor. Thiol patterns on a gold
surface (comprising approximately 1 nm Cr and approximately 45 nm
Au electron beam deposited onto standard microscope slide from
Fisher Scientific) was made using a polydimethylsiloxane (PDMS)
stamping protocol. The protocol employed was optimized to minimize
transfer of material from the PDMS stamp to the surface and to
produce one monolayer of thiol on the surface. All images were
taken with p-polarized light. FIG. 12 shows a series of images
taken of a thiol and water pattern with an optical interference
filter positioned a several different tilt angles. FIG. 12A
corresponds to a center wavelength of 857 nm, FIG. 12B corresponds
to a center wavelength of 852 nm, FIG. 12C corresponds to a center
wavelength of 845 nm, FIG. 12D corresponds to a center wavelength
of 830 nm and FIG. 12E corresponds to a center wavelength of 814
nm. The hexadecanethiol layers correspond to the light regions of
the images and the water layers correspond to the dark regions.
Square regions created by contact with the stamps, approximately
500 .mu.m by 212 .mu.m. As illustrated by FIG. 12A to 12E as the
filter is tilted away from optimal position for this sample, the
contrast between regions of different refractive indices decreases.
As illustrated in FIG. 12A, SPR sensors of the present invention
are capable generating high optical quality images of a probe
region having refractive indices.
An upper limit to the lateral resolution of less than approximately
50 .mu.m was experimentally determined for the exemplary SPR
sensor. FIG. 13 shows images of thiol patterns with minimum feature
sizes of approximately 100 .mu.m (A, left side) and approximately
50 .mu.m (B, right side). The image shows one dimension
foreshortened by a factor of 0.43. In the direction of surface
plasmon propagation, the lower limit to the lateral resolution was
determined to be >50 .mu.m. This is in agreement with the known
surface plasmon propagation length on Au in the near infrared.
FIGS. 14A-D show images generated upon the adsorption of protein
bovine serum albumin (BSA) onto a gold surface. All images were
taken with p-polarized light and a center wavelength of about 853
nm. The image in FIG. 14A shows a small region of the reactor with
a background of water (RI.sub.water is about 1.328 at about 850
nm). FIG. 14B shows an image of the same region of the reactor with
a 2 mg ml.sup.-1 solution of BSA in phosphate buffered saline
(PBS). FIG. 14C shows an image of the same region with a background
of water after pumping water through the reactor to remove all
unbound protein. FIG. 14D shows a difference image resulting from
subtraction of images in FIG. 14A and FIG. 14C. The refractive
index change is due to the adsorption of protein onto the Au
surface (for a monolayer of BSA in water, RI is about 1.331). This
change in refractive index corresponds to a change in percent
reflectivity of about 26%. These measurements show that SPR sensors
of the present invention are capable of the sensitive detection of
changes in refractive index due to adsorption of protein in the
probe region.
* * * * *